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1. Overlay Pew ayn ia Achaea ti Fig 4 61 Image Display window with two tracks plus ratio track Split xy mode 4 78 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 3 Z Stack This function permits a series of XY images to be produced in different focus positions Z slices When the ZStack button is pressed the Z Settings button is automatically activated and the Z Settings panel is displayed in the Scan Control window However it is possible at all times to switch over to setting changing the scan parameters or the PMT photomultipliers and lasers via the Channels and Mode buttons The additional XYscan XYcont Line Sel and Range buttons are available on the right hand OPERATION Acquire Menu Carl Zeiss 74 Scan Control EIEI Line Frame Stack Z Size 19 64 pm Focus 0 00 pm Z Sechoning Mark First Last Hppernine 2 Sechonihg Hum Slices G 4 lt as gt Interval uri 3 93 U ss gt Curent Slice E 4 E Fast Stack Keep Interval Keep Slice side of the Scan Control window and the labeling of the Single button changes to Start Move to First Mid Last rT xvZ tt AL Auto Corr Auta Z The Z Stack function is deactivated by clicking R again on the Z Stack button Z Settings panel overview The parameters of the Z Stack to be created are defined and displayed online in the Z Settings panel Range Fig 4 62 Scan Control window Z
2. 7 9 PINOS eae a aware ansicaen anaeess 4 319 Pinhole adjustment se dyer mauten dieu 4 215 Pixeldep taia r te 4 321 N EE PAON E TEE EET 6 8 control elements ieee 6 9 Preview print wucacbecnctaeaceiunuatact einushennoneucas 4 309 PERG neea r a N 4 34 4 309 Content of display window 666 6 14 Proces Sandee intact tetera ceincesyseadinle 4 15 4 121 E E EAP Aner AIA IEE ATEA 4 121 channel shift enanereeseninokmokiees 4 135 CONTAS eoon ERE 4 132 CODY arr eae 4 127 CUO CATO A ei i 4 128 eee ea SE EN EA ree nee Rn eee 4 129 TET OO VEL C aein eE 4 133 FUTONS hy eee iat omnes Stace sat eet Sia Aenea 4 125 FI SEEE ce tec oocysts E AA T 4 126 SUDICE oee 4 124 Process MeNi ta cteehaectnat a a a 6 5 ARC eee ee ee eee Ce ee 4 261 PPOJACHO MsavenucinGaboraten E 4 155 PROD GCS apisia 6 3 R PAO EET EAEE E PE tances 4 82 Rage Indicato rennon eonna 4 319 4 321 RaO a A AEE N E E 4 126 Ratio Settings 4 64 4 65 7 19 Carl Zeiss Recording configuration 0 4 51 4 60 T poe ea ieee aera neater 4 25 Te 4 118 eens PE nn 9 Fe N 4 241 GUUS EEEE S eae teae is ROJ pe ete Scents A eines een cited ieee Eitan 4 90 4 112 Of aZstack aoa Running down the operating system 4 326 Viti eat ROl aneneen 4 106 S TOODA eree eee ee ae ee 4 15 6 3 6 6 TOD OOKAONY isnan eE 4 278 SEG E E AA onan ciscttacnaisecates 4 243 3D measurement functions 06 4 304 PS EERE Bessa ciatanh E E 4 244 Data
3. Region i Moan Image E Region region Measure s filter ment Image sequence Minimum volume Feature name Measurement condition Feature parameter Fig 6 36 All regions found are checked according to certain conditions The voxel volume of each region must be equal to or greater than MinVolume The measurement condition must be fulfilled Only those regions that meet all the conditions are valid for the measurement The region can be measured or labeled Measurement is a process that produces data Labeling is a process that generates an image volume 03 06 B 45 0019 e 6 51 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan Automatic Object Measurement Object Features A measurement feature describes a region characterized by a number e g volume area or a densitometrical statistic The features can be selected on the Object Features and Volume Features tab sheets dh Automatic Object Measurement General Object Features Volume Features Condition Available Features Selected Features Ellipsoid filled Surface area Surface area filled Sphere diameter Sphere form factor gt Number of holes Mean densitometric Standard deviation densitometric Minimum densitometric Maximum densitometric Select All Remove All Cancel Apply Fig 6 37 The scalings and units are taken automatically from the assigned sequence The measurement features can be selected individually f
4. Landscape ja e Clicking on the Setup button opens the Print Network Cancel Setup window in which you can specify print parameters Fig 4 302 Print Setup window e Click on the slider to change the zoom value of the selected items 4 310 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 26 Display Info This function allows to display the parameters used during image acquisition of the image s displayed in the Image Display window use any image format remove the info display The settings of Chan Zoom Slice Contr and Palette are not relevant for this function In the Options menu in the function Settings with the tab Image Status Display parameters to shown are determined Click on Info will show the parameters Click again to hide the info display convallaria AIM jor x ScanMode Stack Select Display Scaling x 0 20 pm Y 0 20 pm Z 0 40 pm Stack Size x 101 5 pm Y 101 5 pm Z 26 0 pm ScanZoom 23 Objective Plan Neofluar 40 1 3 Oil Average Line 4 Pinhole Chi 79 um Ch2 60 um Ch3 69 um Filters Chi LP 650 Ch BP 505 530 Ch3 BP 565 615 1R Beam Splitters MBS HFT U V 488 543 633 DBS1 NFT 635 VIS DBS2 NFT 545 DBS3 Plate Wavelength 543 nm 100 3 633 nm 36 488 nm 5 User administrator Ready 512 x 512 x 68 3 channels 8 bit Fig 4 303 Image Display win
5. E T Ts je A Fleady 51262006 1 a 1G 2 ehannati 0 bal Flaw imapa data Dinplay Zoom LZ Palita No Palita Fig 4 260 Image Display window Gallery display 4 252 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 20 Display Histo 4 13 20 1 Display Histo Overview This function allows to display a histogram distribution of pixel intensities of an image show the histogram values in table form copy table to clipboard or save as text Tile analyze the colocalization between two channels measure area and mean gray value and standard distribution in an area show separate histograms for each channel in a multi channel image L Colocalization is only available in case of a two or multi channel image The settings of Chan Zoom Slice Contr and Palette are applied Click on Histo will display the Histogram toolbar Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed e Click on the Histo button The Histogram toolbar will be displayed on the right fluo tmre 23 4 9971 AIM Display Absolute Frequency Che 1 60 40 20 oO a 100 150 200 250 Intensity Masimum frequency is 3r at Intensity 53 alues found from Minimum 25 to Maxinurn 226 Absolute Frequency Chl 2 60 40 z0 0 50 100 150 200 eo Intensity
6. E mit Fig 4 6 File menu Mantar Ed F Options TE o Multi Pririt Fig 4 7 Subordinate File menu toolbar 03 06 B 45 0019 e 4 17 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan Create New Database HE 4 4 1 Create New Image Database Create in a Database sE Images File name Convallaria Create Create type Database Files mdb Cancel Fig 4 8 Create New Database window The New function permits the creation of a new image database e Click on the New button in the File subordinate toolbar of the Main menu This opens the Create New Database window for the selection of drives directories and subdirectories e Enter the name of the image database you want to create in the File name text box e g Convallaria e f you want to create the image database in a certain folder drive directory click on the arrow button next to the Create in box This opens a drop down list box showing all folders available for selection To move up one layer of folders click on the EJ button Cancel allows you to stop the process e f you want to create a new folder click on the E button e Click on the Create button This creates the new image database in the selected drive and directory The database files of the LSM 5 software have the filename extension mdb The Convallaria mdb window appears presenting the opened image database with
7. ccccee 4 218 ODJEVEN 4 212 Pinhole adjustment cciastacteddatiacpuaastenums 4 215 REDOO amre siete 4 220 TESCO eenaa e r 4 220 Mathematical morphology ccccccee 6 25 IMIBCTIRSU AST casa since teats erna 4 83 Marker err tote ce cbe ce ssea 4 100 4 103 WIC AM cae euti caesarean ae 4 265 4 312 Mean ROM uriini ee aE 4 106 Measurement COncept snsisissnensieernenn 6 50 Measurement Me nu cccccccececeeeeeeeeeeeeeees 6 6 Measurement PIOCESS cccceeeeeeeeeeeee een eens 6 51 Median filter 4 131 MIIGEOSCOD SE Conto lennin 4 40 LEIE EAEE ENT AEE A 4 71 monochrome image display 005 4 227 MUIT TCE ee a mmee ner eT 4 51 Multiple channel ccccceceeccceeeeeeeee ees 4 322 MUTIRI eenn i 4 60 4 61 N Negation of IMAG eerren 6 39 N a TEETE E EE AEE EE 4 55 Non Descanned i ecnvaieshundetcussladuelecumasies 4 67 O Object features BENSOME VICo E 6 55 GeOMOEU Caaiiis a tala 6 54 ODECE a fadremarie 4 212 03 06 B 45 0019 e List of Key Words Carl Zeiss OC IO EE EE 6 31 ODUON anoe acer 4 16 4 198 Dye Catal ASC nes ere 4 199 ACW AITE astoen rir ON 4 329 SEUNG S erii 4 200 SONAE e e at Ree nena Re 4 327 PCAC AV NoNe E 6 37 Orthogonal SCCHONSincatnccmutrentecoted 4 246 ON peat T E 4 230 P Pale Cii tects 4 237 4 319 4 321 Parocca eaa n a beantabahevaceets 4 214 E E E T A EE AE D E 4 25 Paste Ma Daea 4 146 Piezo fine focusing stage n 7 7 Piezo objective focussing device
8. 3 You can set the x y and Z scales to an identical ratio by opening a context menu in the Image Display box with a click of the right mouse button and selecting the Metric equal ratio function The displayed boxes for rotation angle and relative scaling percentage value z x ratio permit the setting of identical perspectives for different images e g the plot of several topographies 3 The Profiles and Filled 3D display modes permit a color palette e g Glowscale Rainbow or User defined to be loaded or redefined by pressing the Palette button see page 4 237 4 286 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 13 24 6 Context Menu of the 3D Display Mode e Click in the Image Display box with the right mouse button to open the context menu The context menu for the 3D mode currently selected is displayed e Click on the required option with the left mouse button to execute the function 1 Metric equal ratio item This option is available in all of the 3D display modes After activation of the function the x y and z scales are set to an Identical ratio 2 Export profiles item This option is available in the Profiles and Grid 3D display modes Use the function to save the Profiles or Grid data as a text file e Open the context menu with a click of the right mouse button then click on the option Export with the left mouse button The Save As window is opened e Select the director
9. Sl4nm 01 4 gt H seim 0 1 4 gt gt e Select the required laser wavelength and its A gt intensity under Excitation esam o A gt so0nm o A Foo e f required switch the relevant laser to On ki gt Laser button E a a a y e f more than one ROI has been defined under Fig 4 100 Excitation of Bleach Track panel Bleach Parameters a different laser intensity and laser line can be defined for each region Toggle between the buttons indicating the number of the ROI and check the laser line and intensity for each ROI 4 5 8 5 Start End a Bleaching Procedure e The bleaching process will be started via the Bleach button However it is also possible to start the bleaching process via the Bleach button in the Time Series Control Help201 window or to combine it with a time series When a trigger key Is activated to start the bleaching procedure the Waiting for Trigger message first appears in the status line of the Bleach Control window In that case the bleaching procedure is started after activation of the relevant trigger key e The bleaching process can be finished via Stop Help202 in the Bleach Control window 3 Stop does not only stop the bleaching process but the entire scanning process 03 06 B 45 0019 e 4 113 OPERATION Acquire Menu Carl Zeiss 74 Stage and Focus Control X Close Focus Position HA re Focus A work i 0 00
10. The LSM Program is blocked untl this macro is closed Reset window Fig 4 187 LSM 5 LIVE LSM 5 LIVE DuoScan For temporary optimization of the LSM 5 LIVE beampath try to avoid a modification of the general adjustment in the pinhole dialogue Use the Optimize slider instead The general setting of the confocal aperture might still be correct for other scan parameters The Optimize slider can be set independently for each channel The slider is moved to the one or the other side while scanning continuous The brightest image result is the optimal setting This setting doesn t affect the general adjustment of the LSM 5 LIVE collimator and confocal aperture To improve the internal adjustment of default positions of motor driven parts the Reset Servo macro Relnit is available After one hour we recommend to start the Relnit macro to reset all default positions for the actual warm up state of the system Repeat this whenever you experience changes in the system performance After running the macro close the window 4 186 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss 4 8 8 Image Match Scanfield Transformation Macro Adjusts the Image match between two scanning imaging systems or a manipulation and a scanning imaging system To adjust the image match set up a multitracking that creates one channel for each scanning and or manipulation system In case of the LSM DuoScan use a T
11. B 45 0019 e 4 Carl Zeiss 4 I IMPORTANT NOTES FOR CHAPTER 4 B 45 0019 e LSM 5 LIVE LSM 5 LIVE Duo Scan 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Purpose Carl Zeiss CHAPTER 4 OPERATION CONTENTS Page 4 OPERA HON isca a a ag Sg Sg tae eE siete 4 9 4 1 PUTDOSE min a vances 4 9 4 1 1 SO a E E 4 9 4 1 2 Windows and Window Elements essesi e a iaai 4 10 4 1 3 CONVENTION TOF Mhe Text in tms Manual csser heat cee oe eae a ely Siete alone att 4 11 4 1 4 Back UDE nen en Ce ann a Or ete ce RCE em TT nee Sete S eo eee meee eee eee 4 11 4 1 5 SO Wale Oe ia tl Ol ste sarees senate a nsiaga ct santeleatond R adeeb a 4 11 4 2 SWwitching on the SVS CCIM scarce ice eis ccc ees E a aa 4 12 4 2 1 Switching on the HBO TOO messaer kaana huss hata aa aa a a ommend aaa ond nae hone 4 13 4 2 2 Staring ine LOMO Progra sense a a E bated 4 13 4 3 Main Men sonc thas aanceeumeunenceasnccoaane doplcaateacacdvedameceanacceameausmauaiees 4 15 4 4 File Men iieii a a a aS 4 17 4 4 1 Create New Image Databd Enrere a A a eee 4 18 4 4 2 OpEMEX S UNG Mage Dala DIS Orena A 4 19 4 4 3 mage Database VVINGOW oser E EA ened arnadnawennncusaionie 4 19 Aaa F DEMO E E canauaauh aaitne miei tec samen eeeeccets 4 21 AAS 2 Galerne Display ModE ciaciinssentcccancreted ba tenedias a E E E ARA 4 23 AA Tape BD May VOC Cyaan sc eee snc teh aa E ANE 4 24 4 4 3 4 Load an Image into the Image Display WINdKOW ccccccsseececceeeeeeceeeeeeeeeeeaeeeeeesaeeee
12. C Activation of the Ratio channel R1 R2 through assignment of an existing color R1 or definition of a new one Activation deactivation of the Ratio channel via the check box Source 1 ChL1 T1 Selection of the channels of which the ratio is to be formed from the relevant list box Source 2 in ratio settings includes the option to select 1st Image for R1 and or R2 e g to calculate F F for single wavelength dyes A suitable color can be assigned to each of the two Ratio Channels R1 and R2 in the same way as for the photomultiplier channels The channels of which a ratio will be formed are selected via the Source 1 and Source 2 list boxes e Click on the arrow button to select the required channel for Source 1 and 2 from the list box now opened fs The ratio to be formed between the selected channels can be defined more precisely using one of the four preset formulas in the Scan Control window after activation of the Channels button and a click on the relevant ratio button e g R1 for online display of radiometric or single wavelength dyes The Set by min max function in Scan Control window Channel mode allows the definition of the display scaling according to the expected minimal and maximal values 03 06 B 45 0019 e 4 65 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 3 8 Camera Detection Panel Configuration Control Channel Mods The use of this function permits the use of
13. Fig 4 293 Render function visualization aid A zoomed rendering setting permits the zoomed section to be moved via the cursor keys after a click on the 3D window If a change of the 3D image angle follows centration is made on the center again Deleting a Shading Model e Select the model to be deleted in the Shading Model List then click on the Remove button The model is deleted 4 292 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 11 Renderer item This option is only available in the Shaded 3D display mode After selection of the Renderer item the 3D Renderer window appears It allows the selection of the hardware and software option which shall be used for the 3D graphics calculation OpenGI Software The graphics calculation is performed using the installed software OpenGIl Hardware The graphics calculation is accelerated by using the installed graphics processor Direct3D Software Hardware Rasterizer Hardware These options can be used for offline versions of the LSM 5 software for PC s with the WINDOWS 2000 or XP operating system not for WINDOWS NT OPERATION Display and Analysis of Images Carl Zeiss 30 Renderer OpenGl Software GDI Generic Microsoft Corporation Version 1 1 0 Cancel ied GDI Genetic Micrasoft Corporation Version 1 1 0 C Direct3D Software Microsoft Reference Rasterizer Software ertex Processing Version 8 C Direct3D Hardw
14. Info jLow fo Cy High 5 j Table ime secon H lie Show Table Copy Table Save Table l Chi gt Ch3 Ch4 2 Ready 151 x 87 x 8 3 channels 8 bit Display Zoom 1 Palette No Palette Fig 4 304 Image Display window Mean ROI display for time series in single plane B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss The Mean of ROIs toolbar contains the following function elements AOls used during scan opert ROIs selection box Display of the ROIs used during scanning of the time series and of the other ROIs available in the system 2 xl Add button Opens the Add ROI List window for the storage of Add changed or newly defined ROIs under a new name Remove button Deletes the selected ROI from the ROls selection box Bemave Type Position Dimension ROI data Display of the data of the ROI selected from the ROls selection aS TY K Y box On deactivation of the check box of a ROI Its intensity values from Mi CJ 43 152 88 the Intensity Time diagram are not displayed Arrow button Activation of the mouse button for resizing or movement of the ROI in the Image Display window Bezier button Activates the Bezier figure drawing mode The first click sets the starting point each additional click adds a further line a double click on the starting point closes the figure and ends the procedure Circle
15. Store settings of the Bleach Control Proceed as follows to store the entire settings of the Bleach Control window e Enter a name in the Settings list box and click on Store to store the settings If required stored settings for the bleaching procedure can be reactivated quickly e Open the Settings list box with a click on the arrow button and select the required name with a click of the mouse 03 06 B 45 0019 e 4 111 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan e Then click on the Apply button to activate these settings The Bleach Control window will be updated accordingly Delete settings of the Bleach Control Settings which are no longer required can be deleted e Open the Settings list box and select the required name e Click on the Delete button This stored setting will be removed The bleaching procedure can also be activated via keys 1 to 4 of the Trigger Control e Open the Trigger in list box with a click on the arrow button e Select one of the trigger keys 1 to 4 e g Trigger4 It is also possible to trigger an out signal via trigger control Bleach Parameter 4 5 8 3 Bleach Parameter Panel Iterations E The Bleach ParameterHelp193 panel allows you to GT Defne Region determine how often the bleaching process shall be performed and to select the area for bleaching Fig 4 98 Bleach Parameter panel in the scan image via the Define Region button A Ulee Eegi E e Enter the number
16. 4 N D DN ai E DERN cn Diagr in Image 03 06 Arrow selection button Activates the mouse button for selection resizing or movement of the profile line in the Image Display window Resize Click on handle and hold down the mouse button move the handle release mouse button Movement Click on line and hold down the mouse button move the entire line release mouse button Line with arrow button open arrow Activates the straight profile drawing mode Click into the image and hold the mouse button drag a line in any direction and release the mouse button to end the procedure Open polyline button Activates the open polyline drawing mode The first click into the image sets the starting point each additional click adds a further line right mouse click ends the procedure Open free shape curve button Activates the Bezier figure drawing mode The first click into the image sets the starting point each additional click adds a point right mouse click ends the procedure Line button This button allows you to determine the line thickness of the profile line It has no influence on the way the intensity profile is generated Color selection box The colors displayed in the Color selection box can be assigned to the overlay profile line with a click of the mouse The currently selected color is displayed in the larger rectangle top left of the selection box Diagr In Image button The profil
17. 9 EK 4 Program Flow Action Blocks If any action s should be repeated a defined number of times the Repeat block Repeat pl should be set into the loop of the program flow The number of repeats is set in the properties list The repeat is only performed using the connection of the right arrow After finishing the repeats the program flow connected to the arrow pointing down is performed y Using this block in the program flow allows to assign new values to a variable ssignments using mathematical calculations By passing this block the variable will be transiently changed and the new value can for example be used to assign different numbers to the image name or to Increase the time delay during a time series acquisition L The two blocks are used as decision makers Depending on the value of a predefined variable the program flow can be directed to one of the two possible directions The expression of the variable s value which should be used to decide on the program flow is set for each block in the properties list It takes the actual transient value of the variable into account a 7 vee pi k J 4 hai 03 06 B 45 0019 e 4 193 d a Ne 4 Process Action Blocks EED 4 ad OPERATION LSM 5 LIVE Macro Menu LSM 5 LIVE DuoScan This block introduces a delay in the program flow The delay time is started when the block is reached within the program flow The block al
18. All Threshold Ambient Specular Shininess Background Color C Projection View angle Fig 4 275 3D Rendering window e g Surface Advanced rendering mode LSM 5 LIVE LSM 5 LIVE DuoScan 4 13 23 4 Appearance Settings The Appearance button opens the 3D Rendering window This window allows settings for Light Material Background and Projection properties to be defined for all 3D projection modes Depending on the selected 3D projection mode different setting parameters are displayed In the Shadow projection the parameter Roughness is also available and can be set between 0 and 1 A default setting is permanently available for all modes If individual settings for 3D rendering are to be used again they can be stored and loaded when required 3 See also Show Processing Parameters page 4 289 Proceed as follows to save individual settings e Click on the Add to List button e Enter a name in the opening Add Shading Model to list window e Click on OK The settings are saved and the entered name appears in the Shading Model List selection box e To activate the settings double click on the relevant name in the Shading Model List selection box Settings which are no longer required can be removed e Select the name of the setting to be deleted with a click of the mouse in the selection box and then click on the Remove button The setting Is deleted 4 274 B 4
19. Function description Cycle delay or Time List of the stored sets of Cycle Delays or Time Intervals for time series Interval list box Apply button Application of the sets of delays for time series selected in the list box Store button Storage of sets of delays for time series Delete button Deletion of sets of delays for time series from the list box Time buttons Activation of the time for the time series set for the relevant button Time input box Determination of the cycle time for the currently activated Time button arrow buttons slider Unit buttons Selection of time units min sec or ms Trigger in list box Selection of the trigger key 1 4 to be used to activate the Time button for the delay time Trigger out list box Selection of the trigger key 1 4 for the out signal Setting delay time or time interval e The delay time or time interval to be used during the Time Series is set to a default value by activating a Time button For this purpose the relevant time must be assigned to the Time button first e Activate a Time button with a click of the mouse e Set the required delay time or time interval via the slider arrow keys or input box near Time The set time is displayed online on the button Select the time unit by clicking on the relevant button near Unit You can assign different times to all the six Time buttons and store this assignment either as a set of delays or of time intervals e Enter a name
20. The colour can be redefined by clicking on the coloured button on the right of the dialog The standard Windows colour selection dialog is opened The solution is done by clicking on one of the colours or by entering appropriate numbers in the corresponding edit boxes Pressing the OK button will close the colour selection dialog and update the Display window immediately Only those channels which are available in the image sequence can be defined Parameters Image Image sequence to edit Weight Colour weighting for each channel Colour Base colour for each channel 6 10 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss 6 3 Functions 6 3 1 Functions in the File Menu Open Image This function reads a Zeiss LSM Ism Zeiss LSM TIFF 000 tif or Carl Zeiss Vision O img image sequence from a disk or network drive Open Image x Files CALS ME1053DforLemslmagess Mitosis 44 07 6 TIF File Fath W Preview jaan ETIF CNLSMS1 0 3D forLem Images Mitost EJE EJ LSM510 EJ 3DforLsm J images Files of Type 512 512 15 3 Ch fall TIF Images tif T 4 Open Cancel Fig 6 6 The individual files of a Zeiss TIFF image sequence are read and saved as an image sequence in image memory In addition the image properties are read out of the TIFF files and allocated to the image sequence Input The directories of the current drive are listed in the Director
21. e Click on the arrow button The image selection box is opened and all the currently loaded images stacks time series with a Lambda dimension are displayed in a minimized form e Click on the required image This image will then appear in the display box of the image selection box and has been selected 4 6 11 3 Definition of Channels Panel In the Channels panel the number of reference spectra number of fluorescence channels can be selected from the channel selection boxes e Select the references fluorescence dye spectra which are present in the sample with the check boxes e f necessary change the colors of the relevant fluorescence channel e Anew window with the resulting channels of the unmixing procedure opens immediately There are several settings that can be chosen for linear unmixing Autoscale balances the intensity of the unmixed images Generate channels with Residuals shows the difference between the offered spectrum and the resulting Image in intensity values The higher the intensity in this additional channel the worse the fit of the spectra to the dataset This occurs for example when pixels are saturated and indicates to repeat the image acquisition with different settings to avoid overexposure or in extreme cases to chose different reference spectra Ask for Background ROI when this is checked a window appears before the calculation starts which asks for the spectrum of the background which is then subtracted t
22. 1 10 4 sw 40 SO BO 7074 Increment walt Time 0 1000 Fig 6 4 The image sequence is displayed in the Display window The display process is working as a background task other functions can be executed while the player is running There are several ways to stop the player by closing the player window by pushing the red Stop button of the player window the window remains open by closing the image window The Increment parameter specifies whether each sequential image 1 should be displayed or whether some sequential images should be skipped during display The value 2 skips one image for every sequential image displayed in other words it displays only every second image The parameter Wait Time states the delay in milliseconds between two successive sequential images The maximum display speed depends mainly on the hardware The sequential images are always displayed in their entirety regardless of the set delay 6 8 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan User Interface Carl Zeiss Control Element of the Player The three arrow shaped controls on the scale show the start slice and the currently displayed sequential image The values positions can be changed using the mouse Press and hold the left mouse button and move the pointer to the desired position The set values are shown in the numerical windows at right E Start slice Currently displayed sequential image End slice The
23. Activation deactivation via check box of the selected channel ChL 1 ChL 2 for the scanning procedure by assigning an existing color icon or defining a new one a CO 03 06 e Select the appropriate filters and activate the channels e Click the Excitation button to select the laser lines and set the attenuation values transmission in in the displayed window For the configuration of the beam path please refer to the application specific configurations depending on the used dyes and markers and the existing instrument configuration e Clicking on the Config button 4 Track Configurations ue opens the Track Configurations Store Apply Configuration window Fig 13 to load store or T delete track configurations CFP CFPAYFRP FRET Cys e For storing a new track configuration enter a EEn desired name in the first line of the d Configurations list box and click an Store e For loading an existing configuration select it in the list box and click on Apply Fig 13 Track Configurations window e For deleting an existing configuration select it in the list box and click on Delete Setting for multi track configuration in Channel Mode The Multi Track function permit several tracks to CAA be defined as one configuration Channel Mode es Configuration for the scan procedure to be EAE ONE eee stored under any name reloaded or deleted Single Track Multi Track Li
24. E TineSeree Eateach stage m ra Pa Al J D iae Heo Cog Scen Edna Fig 4 91 EditBleach Button Hence the Define Region button only allows to select the upper and lower border of a bleach region It is possible to select more than one bleach region Bleach Regions are drawn directly in the image window The bleach wavelength is choosen in the Excitation laser wavelength menu on the bottom of the bleach control dialogue Check the required line and set the power with the sliders A single bleach event can be started interactively by pressing the Bleach button Fig 4 92 Stripe Bleach Function 4 108 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Ready G12 512 2 charret tal Fig 4 93 Image Window Bleach series To set up a time series with a bleach event the bleach region is selected as described above In the Time Series window a time series is set up co Lpa SS LIWE fie Agaa Foe vey Midi eine Fie pa i ai acns EP St Wiri Hizo ii Mariam Wii rep V1 iu Config A Laver AGIA Eda FOI Trab ene of Ect beck Shape Tt Suh Fig 4 94 Time Series Button 03 06 B 45 0019 e 4 109 Carl Zeiss Settings Apply Store Delste Bleach after E number scans Close Bleach Bleach repeat after E EAC nu
25. Masimum frequency is 62 at Intensity 39 alues found from Minimum 18 to Maximum 136 fF eady F124 2008172176 2 channels 3 bit Raw image data Palette No Palette Fig 4 261 Image Display window Histogram display 3 The Histo button can also be used online during scanning 03 06 B 45 0019 e 4 253 Carl Zeiss Histogram functions Skip Black button Skip White button Statistics Step input box Show Image button Show Table button Copy Table button Save Table button Area functions Area button Save Values button 4 254 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Ignore black pixels gray value 0 in the histogram Ignore white pixels gray value 255 or 4096 in the histogram Displays statistical parameters Mean Intensity Standard Deviation Number of Pixels and Size of Area in a additional table Area measurements of very small areas lt 10 pixels give only approximate values Set the number of intensity steps which shall be displayed in the diagram Step 1 corresponds to 256 intensity steps Step 64 to 4 intensity steps in case of 8 bit images Reduction is made by averaging Shows the image in the Image Display window beside the histogram The histogram is shown as a table at the bottom left of the Image Display window The histogram table is copied to the clipboard The histogram table can be stored as a text file extension txt Interactive
26. OPERATION LSM 5 LIVE Display and Analysis of Images LSM 5 LIVE DuoScan 4 Export x y z triples item C helpfiles LSM 510 Rel 2 02 E Lem510_2_3 R LSMIB_READM LSMI_READM This option is available in the Profiles and Grid 3D display modes Use the function to save the Profiles or Grid data as a text file e Open the context menu with a click of the right gt mouse button then click on the option Filename test tt Export with the left mouse button Save as Ippe Text Files tut Cancel Fig 4 2837 Save As window x Y Z 0 00 127 0 00 10 55 1287 57l 2110 1287 723 31 65 1287 702 42 19 1287 399 52 74 1l2e7 3 19 Fig 4 288 Topography table 5 Copy x y Z triples to clipboard item The Save As window Is opened e Select the directory where you want the text Tile to be stored enter a file name and click on Save A text Tile containing the topography in the form of an XYZ table is generated This option ts available in the Profiles and Grid 3D display modes After selection of this option the Profiles or Grid data are copied as an XYZ table to the clipboard and can be inserted in other programs using the Paste command iS 4 288 Please make sure that the amount of exportable data is adequate to the maximum importing size of the following software package To lower the amount of data points use the profile distance slider B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE
27. Or e Click on a Gauss or Average sub button and select the required degree of smoothing from the selection box with a click of the mouse FFT button This function performs a Fast Fourier Transformation FFT in the frequency range applies highpass or lowpass filtering in the frequency range and performs the inverse FFT e Click on the FFT button the FFT Filter window opens e Click on the arrow in the filter Type select box to choose an adequate filter function Gauss Lowpass Gauss Highpass Butterworth Lowpass Butterworth Highpass e Select a position of the Cut off slider to display either the lower frequencies waviness with the lowpass filters or the higher frequencies roughness with the highpass filters e The Cut off frequencies ranges trom 1 1000 of the X dimension of the stack to four times of the X dimension of the stack The dimensions of the filtering is given in units of um e Select a position of the Degree slider The filter functions can be calculated from 1 order to 5 order accuracy e Click on the Close button closes the FFT Filter window 4 282 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 2 Changing the topography geometry The three buttons in the Geometry button bar allow the surface geometry to be changed Inverse button Inverse surface Depths change to heights and vice versa Fit button The following fit modes can be set
28. Selection of the laser to be switched on off and setting of the laser output is performed in the subordinate setting panel Laser settings panel lower Switch on off the required laser or set Standby operation Display Maximum Power Wavelength and Status of the relevant laser 4 5 1 4 Settings e Click on the desired laser on the upper Lasers panel This highlights the selected laser On the lower panel of the Laser Control window activate the laser as follows This applies to the 405 Diode laser 405 nm e Click on the On button directly not on Standby This applies to the Toptica and the Sapphire lasers 635 488 nm e Click on the Standby button Wait for the laser to heat up until the Status ready Standby message appears approx two minutes e Click on the On button Status ready On appears This applies to Compass 315M laser 532 nm e After selecting the laser click on the On button The required laser for image acquisition is now available fs After switching on the lasers in the laser control window and their status ready the system can be used for imaging However for quantitative imaging it is recommended to let the system warm up for 40 minutes This applies to the Coherent UV laser 653 II Enterprise and the Ar multiline laser e Click on the Standby button Wait for the laser to heat up until the Status ready Standby message appears approx two minutes e Cli
29. The extent of the data displayed in the Scan and System Information window depends on the settings made in the Options menu under Settings see page 4 200 function opens the Scan Information in which the current scan data are e Open the Window menu e Click on the Scan Information line The Scan and System window will be displayed Information Click on the X button to close the Scan Information window B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Help Menu Carl Zeiss 4 12 Help Menu The Help menu permits activation of the Help function and of a window containing information on the installed software version 4 12 1 Help e Open the Help menu e Click on the Help line to open the online help 4 12 2 About e Open the Help menu Carl Zeiss e Click on the About line to open the About Cae window Copyright Carl eis GmbH 1997 2005 i A E in collaboration with EMBL Heidelberg Germany fee The About Wi ndow incl udes im porta nt Portions Copyright 1996 Microsoft Corporation All rights reserved information about the software such as the Version Info Dongle Number 8368 software version number copyright version eee Fae aaa numbers of the various program components and Software option Macro RecordE dit available i Sotware option Physiology available firmwa re d nd the Dong le number Software option Topography available Software option 3D for LSM available Software option 3D i
30. The image can be displayed in Tull size one pixel on the screen represents one pixel of the image or in a zoomed size To zoom the display view click and hold down the right mouse button on the window border and resize the window The aspect ratio of the image will not be changed Clicking on the button el resets the Display window to a full size view of the image see above The title bar shows the currently displayed sequence name The status bar displays the size of the current sequence and the selected slice on the left On the right the cursor position within the window and the corresponding intensity grey value of each channel is shown The Display window can be closed without any effect to the image processing functions If no Display window is opened select the entry Display in the Window menu The scroll bar at the lower right of the window enables to show the images in a sequence The range reaches from one to the maximum slice provided by the current sequence To start the automatic animation of an image sequence start the Player tool by clicking on the button is The colour selection for the channels can be activated by clicking on the button By A colour image can be displayed as a grey value image by clicking on the button mm 03 06 B 45 0019 e 6 3D FOR LSM LSM 5 LIVE Carl Zeiss User Interface LSM 5 LIVE DuoScan Player This function plays back the sequential images of an image sequence w Player E
31. Wrap 2 Clip 3 Normalize 03 06 B 45 0019 e 6 17 Carl Zeiss 3D FOR LSM Arithmetics Subtract This function subtracts two image sequences The Subtract tab sheet of the Arithmetics dialog window must be selected Functions Arithmetics Add Subtract Input 1 a annee Input 2 kooo AY ES Output BE News i Normalize C Shift4Clip Fig 6 12 Cancel Apply LSM 5 LIVE LSM 5 LIVE DuoScan If one or both input sequences are multichannel sequence any number or combination can be selected The number of selected channels for Input 1 and Input 2 must be the same They will be combined from left to right This function subtracts the two image sequences Input 1 and Input 2 voxel by voxel and generates the image sequence Output Note that a resulting grey value may be less than O The parameter Mode determines how a range overtlow negative values is handled 1 Wrap 2 Clip 3 Normalize 4 Shift Clip Parameters Input 1 Input 2 Output Mode 6 18 No normalization the grey values are displayed modulo 256 4096 If the result Is less than O the value 256 4096 Is added to it Negative values are set to O The resulting grey value range is scaled to the range 0 255 0 4095 128 2048 is added to the difference then negative values are set to 0 Values greater than 255 4095 are set to 255 4095 First input Image sequence Second input image sequence Output Image sequen
32. cece ecceeeccccccee ee eeceeceeeeeeeeeeeeesaneeeeeeeeas 7 3 7 2 2 LSM 5 LIVE 2 Channel Biomedical ConfigurationS ssssiisisessiiniuessrririserrrirreerrrrrrernrrrrn 7 4 7 3 Changing Filter lin the 5 Cala nno MOA Eiana G 7 5 7 4 Detaching Attaching the Scanning Module from to Microscope Stands 6ccceee 7 5 7 5 Hints on the Use of the Piezo Fine Focusing Stage anak caserseieeawe ieee cevaneseihaieceitecelokts 7 7 7 5 1 Ganaa DES iDO eaa na a A a T 7 7 7 5 2 APO TUOTA I a T O O naa 7 7 7 5 3 Additional Information on the Operation ccccccceccccceeeecceeeeeeeeeeeeeeeeeseeeseesaneeeeeaaneeeenaanes 7 7 7 6 Piezo Objective Focussing Device cceccccceeecccce ee eeeeeese ee eeeeeeeeeeeeeeeeeeeeseaeeeeeeeaeeeeeeuaneeeeeanaes 7 9 7 7 Specifications of Trigger Interface LSM 5 LIVE eeessinsessiinnuessririrresrrirrrerrrrrrirerrrrrrrerrrrren 7 10 7 8 AxioCam High Resolution Digital Cameras ccccccccceceeccccee ee ceeceseeeeeeeeeeeeeceuaeeeeeeeaneeeenes 7 13 7 8 1 Microscopy Camera AxioCam MRM REV 2 iittadcicen resend ste a dite caauilicseudela duane caverns Sw idedad aadladiess 7 13 7 8 2 High Resolution Microscopy Camera AxioCam HRM ReV 2 cccccccccceseeceeeeseeeeeeseneeeeeeas 7 14 7 8 3 High Resolution Microscopy Camera AxioCam HRC sss usssinissiriuesrirnesririerrrrreerrrreerrrrns 7 15 7 8 4 Microscope Camera Port Adapters for the AXIOCaM ss essiiessrireesrrrresrirerrrreerrrreerrrree 7 16 7 9
33. i ESEA EE Fig 4 118 Filter List and Edit panel Lowpass Kemel Size C 3x3 f 525 TRT User defined L Divisor fi L Factor 2 ARAA EES EEE Divisor 12 2 2 2 A 2 Offset sce ARAA Fig 4 119 Filter List and Edit panel Sharpness LSM 5 LIVE LSM 5 LIVE DuoScan 1 Kernel size The size of the filter matrix can be modified here The effect of a filter increases along with the matrix size However this also increases the time required for filtering e Select the required matrix size by clicking on one of the selection buttons 3x3 5x5 or 7x7 2 Lowpass filter With the lowpass filter the gray value of each center pixel is replaced with the average value of the surrounding neighbor pixels The viewed neighbor pixels are defined by a square The moditied pixel now is the center pixel of the filter matrix Image noise will be reduced by the application of the lowpass filter The cutoff of regions will blur Local maxima will be flattened The dynamic range will be reduced considerably This filter permits the matrix size to be modified only in the 3 preset steps 3 Sharpness filter With the sharpness filter the original image is filtered with a lowpass filter first The result of this filtering is then subtracted from the original image This will improve image sharpness The matrix size can be modified in the 3 preset steps Furthermore divisor values ranging trom 1 to 78 can
34. zi 16 22 6 ai Sa Difference Angle a Panorama M Preview Slice Fig 4 155 Stereo Images window 03 06 B 45 0019 e 4 159 Carl Zeiss OPERATION LSM 5 LIVE 3D View Menu LSM 5 LIVE DuoScan 4 7 3 3 Stereo Images Panel e In the Stereo Images panel set the parameters needed for stereoscopic viewing Mode Basic Angle Right Left Angle Number Images and Difference Angle in the Projection tab and the Mode parameters in the Transparency tab 1 Projection tab Mode Red Green Image Mode Split Images Basic Angle Right Left Angle Number Images Difference Angle This displays a stereo image for red green anaglyph observation using red green spectacles This displays a pair of stereo images for observation through a stereoscope Colored stereo images are also possible Direction angle at which the specimen is viewed 0 trom the front 180 trom the rear Angle between right and left red and green image Number of 3D images slices Angle increment of a sequence Mode Red Green Image Split Images Basic Angle a a Right Lett Angle 12 El L Mumber Images 2 Re 45 18 9 Difference Angle 0 2 SEEE Fig 4 156 Projection tab L gt The number keys permit the direct selection of preset values for Number Images and Difference Angle If the Panorama button is pressed a panorama sequence of the image series is computed 4 160 B 45 0019
35. 240 V AC 10 50 60 Hz 1 phase at 208 V 34 Amps 230 V 31 Amps 240 V 29 Amps 7000 VA max 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Notes on Handling the Laser Components Carl Zeiss 1 8 Notes on Handling the Laser Components My The LSM 5 LIVE are laser hazard class 3B instruments This moderate and high risk class embrace medium power and high power lasers You must take care not to expose yourself to the radiation of such lasers In particular never look into the laser beam Only personnel which has been instructed on laser safety is allowed to operate the system The following laser types are currently intended for use in the LSM 5 LIVE The use of any other lasers as the ones listed below is not authorized 1 Diode laser 405 nm 2 Diode laser 440 nm 3 Diode laser 488 nm 6 Ar laser 351 364 nm UV for DuoScan only Laser type class 4 if mounted on laser module with fiber output class 3B IS Please note that for the maintenance of the UV Laser it is recommended to run the laser at maximum power once a day if the laser is not used frequently or only at low power levels This enables the Autofill process which keeps up the correct tube gas pressure This operation prolongs the life time of the tube and prevents complete tube failure if the laser is not used for a prolonged period of time For details please refer to the Operator s Manual of the UV laser IS Please contact Carl Zeiss if you intend
36. Channels Zoom Slice Overlay 03 06 B 45 0019 e 4 207 OPERATION LSM 5 LIVE Carl Zeiss Options Menu LSM 5 LIVE DuoScan 2 Palettes Color a Switch to mono on activation of a palette If this check box bl is activated the Mono chrome image display mode is switched automatically when a palette is selected in the Color Palette window b Switch to color on deactivation of a palette If this check box bl is activated the Color image display mode is switched automatically when a palette is deactivated in the Color Palette window c Remember color Palette If this check box bW is activated the consecutive image will be displayed using the same palette as the previous image d The background color of images and diagrams can be chosen when clicking on the respective color bar srtteenssnensennsnneesennssnnesensssenssennsssenssennsssneeey Program Stat Hardware Image Display DO an j 4 9 2 17 Save Image files to subdirectory of the database Database and image files to the same directory The Save tab permits the presetting for the Belt peandloprireenterp a r e ahaa storage of scanned or processed images to be changed V Save prompt at closing modified windows Warning before overwrite existing recordsets Activation of one of the three option buttons V Remember Name Description and Notes in the save dialog enables you to determine the database directories to which stored images are assigned Fi
37. Click on the appropriate mark to select it for operation 4 116 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Move To button Clicking on the Move To button moves the stage to the position selected before from the Marks selection box Remove The Remove command enables a selected position to be deleted trom the table The position then also disappears from the specimen carrier display ms The selected position is deleted the position with the next number in sequence moves up one number Remove All The Remove All command deletes all the entries marked in the current session Speed selection box Clicking on the arrow key displays the table of the available speeds for stage movement Click on the appropriate speed to select it for operation Zero button Zeros the Current Position display and thus sets the currently set stage position to O in relation to X and Y The already marked object areas thus receive new X and Y coordinates Mark Pos button Mark Pos allows the Current Position to be marked This marked position Is then stored in the Marks selection box in sequence The marked position is shown on the specimen carrier with a cross and its ordinal number HRZ Zero button Zeros the Current Position display and thus sets the currently set stage position to O in relation to X and Y The already marked object areas thus receive new X and Y coordinates 03 06 B 45 0019 e 4 117
38. For a description of the toolbars Select and Display see Display and Analysis of Images page 4 224 The name of the image is displayed in the Name input box of the Image Database window e f you want to change the name click on the input box and enter the new name directly via the keyboard Faa 120512 466 a e The Description and Notes input boxes allow you to Fig 4 11 Opened image displayed in the Image Subsequently add descriptions or special notes on the Display window recorded image via the keyboard The acquisition parameter settings of the image are displayed in the Acquisition panel Changes to an original scan image are automatically recorded in the Processing panel under History If for example the image was added to the database via the clipboard the entry Imported file will be shown under History Under Image Size the size of the image in pixels for XY Z and the number of used channels are displayed 4 22 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss 4 4 3 2 Gallery Display Mode e Click on the Gallery button All images of the image database e g Convallaria mdb image series are shown in a tiled arrangement of thumbnails on the screen amp Convallaria_mdb AlM Record 5 of 5 Convallaria 6 bit Bl2 512 50 Cone Cornv b Cone 6 bit 8 bit 6 bit Alex Sle alex ble Alex Sle alex 412 Delete Total 1 471 MByte Selected 145 457 kByte Curren
39. LSM 5 LIVE DuoScan 4 13 7 3 Spline The intensity is set in the Intensity Screen via a freely selectable number of knots which permits the creation of an intensity line in the form of a spline The number of knots can be selected from the Number of Knots selection box 3 The original line form is reset via Reset The line form will be retained even when the additional functions are no longer displayed or when the Brightness and Control window is closed 4 13 7 4 Gamma The intensity is set in the Intensity Screen by varying the gamma curve clicking and dragging with the mouse or by moving the Gamma slider It is possible to set gamma values between 0 1 and 2 0 f The original line form is reset via Reset The line form will be retained even when the additional functions are no longer displayed or when the Brightness and Control window is closed 4 236 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 8 Select Palette This function allows to change the palette used for displaying the image s define and save new palettes delete palettes by removing them Click on Palette will display the Palette toolbar Any changes done with this toolbar are effective immediately The standard palettes No palette Range indicator Glow Scale and Rainbow are system palettes and can not be deleted The Range indicator palette is useful to optimize the gain
40. Line button Frame button Z Stack button Z Settings button Close button New button Find button Fast XY button Single button Stop button Cont button Finish button 03 06 When the button is activated the following panels are available for the setting of the scanning parameters for the line and frame modes Objective Lens Image Size amp Line Step Factor Speed Pixel Depth Scan Direction amp Scan Average and Zoom Rotation amp Offset When the button is activated the Channel Settings and Excitation of Track panels are available for the setting of the channels and the laser excitation Activates the Line scan mode Activates the Frame scan mode Activates the Z Stack scan mode display of additional buttons on the right hand side of the Scan Control window When the button is activated the Z Settings panel is available for the Z scan parameter definition The Z Stack scan mode must be active Closes the Scan Control window Opens a new Image Display window Automatic optimization of image brightness and contrast The settings for the Find function can be varied as required using the Maintain menu Continuous scan with high speed This function should be used to a limited extent and only for a short period of time Fast XY switches temporarily to 512 x 512 frame size Single scan named Start in the Z Stack mode Stops the current scan procedure no matter in which window the button is pressed a
41. O e Click on the Config button in the Acquire ee Ee ere Name Channels Light nm F Track subordinate toolbar of the main menu The Configuration Control Help92 window Mies opens l Add Track Store Apply Single Track Config e Activate one of the Single Track or Multi Track buttons and click on the LSM 5 LIVE or Sea rie air andi hanini t LSM DuoScan button LSM DuoSean LSM 5 LIVE Camera The Beam Path and Channel Assignment panel for camera detection is opened ms If LSM 5 LIVE and LSM DuoScan tracks are mixed the active detection port of the microscope has to be set according to the first track A Excitation Specimen Fig 4 48 Configuration Control window LSM 5 LIVE Duo Scan activated 03 06 B 45 0019 e 4 67 Carl Zeiss 74 Scan Control Objective Plan Meotluar 10s 0 3 Frame Size ie 256 s2 768 102 Ale a plz Format 512x 512 kel Scan Speed 41 FPS E Par Fisel Time 30 00 psec Scan Time 24 39 msec Pixel Depth Scan Direction amp Scan Average Data Depth Bit 412 Bit Mode Frame i Method Mean scan Direction oi p Uum GE 1 Zoom Rotation amp Offset Offset Offset 0 00 um Offset 0 00 um Fig 4 49 Scan Control window Frame OPERATION Acquire Menu Single LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 4 Scan Control The scan parameters for image acquisition are set in the Scan Control window The m
42. The Objective button is activated in accordance with the presetting and the Objective panel is displayed in the Objective Control window e Click on the graphical button of the relevant nosepiece mount Position The Change Objective window appears All available objectives are listed in the Potential Objectives directory of the Change Objective window e Select the new objective by double clicking from the list in the Potential Objectives directory e Click on Close to exit the Change Objective window 1 Add Objective This function permits new objectives to be added to the database For this proceed as follows e Click on the Add Objective button on the Change Objective window The Create new Objective window is opened 4 212 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan e Enter the data of the new objective in the Create new Objective window then click on the Apply button The new objective is stored in the database and included in the Change Objectives window You can now activate the new objective as a favorite objective using the procedure described above iS If you have activated the Non Zeiss check box objectives from other manufacturers can also be included in the database 2 Remove Objective You can only remove objectives in the User Defined Objectives directory OPERATION Maintain Menu Carl Zeiss Create new Objective Producer Carl Zeiss Jena GmbH Non Zei
43. a F YolSurks dice oloun ofolume of olurnie ofS urbAres eae es Volume Features ez 1060 39465437 50 d 10460687 50 OF Cancel i Fig 6 40 The regions must be defined by an image sequence Mask Image the objects must be separated trom one another by black voxels with the grey value 0 This sequence is generated with the function Segment If it is a multichannel sequence a single channel has to be chosen The image Dens Image is needed for the measurement of the densitometric features Image sequence properties like scaling and unit are taken from Dens Image A single channel of this sequence if it is multichannel must be selected with the buttons to the right of the parameter The measurement results can be stored to database files These files are tab delimited ASCII files which can be easily imported to major Windows programs like text processing or spreat sheet application Writing database files are independently supported for object and volume features Activating the corresponding check boxes enables it The name of the database is defined with the field Database The files will be located in the subdirectory DATA of the main installation directory The filename extension TXT will be added automatically If the check box Label is activated a single channel sequence will be generated It contains all the measured objects each object is coloured homogeneous but in different colours To copy all measurement values to the cli
44. action block Property Value D Projekte Release 3 5 RTAS WScripting test 2 mdb Name Cell culture Index Description Notes Compress Yes Fig 4 195 Property list of the Append to Database action block Property Value Mode Relative Position Distance i pm 10 Distance r pm 10 Fig 4 196 Property list of the Move stage action block 4 196 B 45 0019 e Property Value Configuration Type Single Track Configuration 496 FITC Load Speed No Load Bits per Sample No Load Pinhole Diameter No Load Gain and Offset No Fig 4 193 Property list of the Beam Path action block The database the images will be appended to Is automatically displayed when highlighting the Append to Database action block The last one used will be taken To update this open the desired database and use the Read back function Type in a name for the images with or without quotation marks All images will get the Same name then For numbering the images use quotation marks for the name and add the variable Index which has been assigned with the value 1 This value or the transiently changed value of the variable will be added to the name of the image The movement of the stage is defined highlighting the Move stage action block choosing the Mode Relative Position in the Property list and typing in the value 10 for X and Y which then will move the stage 10 microns into X and Y direction starting with the current stage pos
45. and processing information to be stored in an image database In the Options menu in the function Settings it is possible to define an Autosave function When Autosave is off the Save dialogue is the Save As dialogue Proceed as follows to save an acquired or an edited processed image e Click on the Save or Save As button in the File subordinate toolbar of the Main menu The Save Image and Parameter As window appears on the screen mr Save Stores a newly created or changed image Newly created images must be given a name and assigned to an existing or new database Save As Stores a previously stored and called up image under a different name If images are called up and stored again the original data and time display will be retained Clicking on either of these buttons opens the Save As window to create and open an image database When the Compress Files W check box is activated the images are stored in a compressed form e f necessary enter a description of the image or comments on it in the appropriate text boxes e The default display in the User text box is the name of the logged on user If you want you can enter a different user name for the current image e Click on the Open MDB button if you want to open an existing image database in which you want to save the current image Click on the New MDB button if you want to create a new database to save the current image Save Image and Parameter As Hame Co
46. and text can be entered via the keyboard The Font button enables you to select the font style and size in the Font window The entered text will be displayed in the left upper corner of the Image Display window after clicking on OK and can be moved to the required position using the mouse The Text window can also be activated with a double click on a created text box and the entered text can be edited subsequently Insert opens up a further window which allows you to annotate coordinates time and Z position with either automatic or user definable units and precision This annotation is updated during image acquisition and can be exported with the image The annotation can be stamped into already existing images Recycle bin button All the overlay elements and dimensions dragged to the scanned image are deleted If one overlay element was marked before this element is now deleted from the scanned image Multiple button On activation of this button the overlay function subsequently selected is performed several times in succession without the need to activate the function button again This function remains selected until the Multiple button is deactivated again Measure button Measurement of the overlay element in the Image Display window On activation of the Measure button the selected overlay element and all the elements created afterwards are measured and assigned with a measuring value The measuring value can be shifted with
47. computer must not contain any data storage item e To switch on the system completely put the Components switch also to ON Now the complete system is ready to be initialized with the LSM Software e After switching on the computer type in the user name and password to log on to the computer e After entries confirm by clicking the OK button or Fig 4 1 REMOTE CONTROL switch Enter The WINDOWS XP operating system desktop appears on the screen showing a number of icons Change Filters icon The Change Filters tool is used to update the filter data in the software after a Change Filters l change of filters in the reflector turret See printed manual CHAPTER TOOLS Stand Select icon Stand ae The Stand Select tool permits a new or updated database to be assigned to the LSM 5 software program This function should preferably be performed by authorized service personnel See printed manual CHAPTER TOOLS re LSM 5 LIVE icon gei cee Start the LSM 5 software program for the LSM 5 LIVE laser scanning microscope 4 12 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Switching on the System Carl Zeiss 4 2 1 Switching on the HBO 100 e If the HBO 100 is required switch it on via the toggle switch of the HBO 100 power supply 4 2 2 Starting the LSM 5 Program The LSM 5 software program can be operated in two different modes with or without connected instrument system In the on line mode
48. e Select the relevant stand icon from the Icon list box and click on OK The Select Name window is closed and the desktop icon is updated e Then click on the OK button in the Select Stand Database window to accept the new settings and to close the window Clicking on Cancel will cancel the procedure After the next restart of the LSM 5 LIVE software program the new database will be automatically read in 5 4 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan TOOLS LSM Image Browser Carl Zeiss 5 3 LSM Image Browser The LSM Image Browser permits images to be loaded imported exported and printed quickly without having to open the LSM 5 LIVE software The LSM Image Browser can be used without dongle When images are opened image processing functions of the LSM 5 LIVE software are available to a limited extent Chan Zoom Contr Palette Copy Save Save As xy Split xy Prev Info e Click on the LSM Image Browser icon on the desktop of the PC The Zeiss LSM Image Browser main menu is opened i Zeiss LSM Image Browser File View Process Options Window Help Fig 5 6 Zeiss LSM Image Browser main menu The following function buttons are available New button Open button Save button Save As button Import button Export button Full Screen button Multi Print button RAM button DISK button Exit button Opens a new database Opens an existing database Saves the current image Saves the c
49. icon activate the 532 nm Line active Transmission eo laser line by clicking on Line Active W If Power C sanm a1 gt o Clase required deactivate other laser lines which are Wo 53 mim 30 4 j e not needed C enm a1 Ha A e Use the Transmission slider to set the laser intensity to approx 30 at Tirst The Beam Path and Channel Assignment panel displays the current configuration loaded ms The set laser intensity must be Fig 4 306 Excitation panel Subsequently optimized for the current situation via the Transmission slider For overlaying fluorescence and transmitted light images activate a second channel without a laser line but the Halogen illumination of the light microscope as the transmitted image light source 3 Choose the BP 675 725 in the emission filter menu and swing in the according red filter at the condensor Of course all other transmitted light applications like phase contrast differential interference contrast DIC polarization contrast Pol darkfield can also be performed 4 316 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan e In the Main menu click on the Scan button in the Acquire subordinate toolbar This opens the Scan Control window e Click on the Mode button e Fora frame scan click on the Frame button e On the Objective Lens Image Size amp Line Step Factor panel select Objective and Frame size for th
50. pairs 0 0 1 1 2 2 with the same intensity can occur Differences between the images cause an irregular distribution in the scatter diagram 007 GFP_YFP une AIM Scatter diagramm Select Dipy Histoyam aktahor Show Table vate Irteretaly GFF 4000 0 00 1000 1500 2000 2500 W00 300 4000 niert GFP cij 100 150 Aipha Frequency Fig 4 263 Image Display window Colocalization display 03 06 B 45 0019 e 4 257 Carl Zeiss Function elements OPERATION LSM 5 LIVE Display and Analysis of Images LSM 5 LIVE DuoScan The following function elements are available in cake Cut Mask panes Channel Toggle Phe Invert Mask Source 1 CHS1 T1 Source 2 ChS2 71 Mask RGE Drawing tools Save at drawing tools 4 258 Displays the scatter Histogram of the two image channels Adds display of the according data table Adds display of the image Displays a movable crosshair for different areas in the scatter histogram Opens the Intensity Threshold window to sets threshold for colocalisation in the scatter histogram Set from Image ROIs button Sets background threshold from ROI Threshold button Cut Mask button Channel Toggle button Invert Mask button Inverts the mask or the scatter diagram Source 1 selection box with Color selection box Selection of the first channel to be selected via the selection box assignment of a defined color via the
51. positions A and B Focus Position 0 00 porn 0 00 um Pressing the Move A and Move B buttons permits 7 l nable test E the defined Z position to be directly approached The Enable test check box permits simulation of Fig 4 69 Z Brightness level control panel the value changes for Detector Gain Ampl Offset Ampl Gain and Attenuation in the Scan Control window without the scanners being in operation ms Ifa Z Stack is performed and the Auto Z Brightness Correction window is opened this correction is automatically performed equal whether the Enable test box is enabled or disabled e Use the focusing drive to set the Z position where the brightness level correction is to be started e n the Scan Control window set the initial values for Detector Gain Ampl Offset and Ampl Gain If required start the continuous scan procedure for this purpose Click on the Set A button e Use the focusing drive to set the Z position where the brightness level correction is to be ended e Set the end value for Detector Gain Ampl Offset and Ampl Gain in the Scan Control window Click on the Set B button e f required check the change of the set values by activating Enable test After the start of the scan procedure the brightness level values are linearly interpolated between the defined references A and B LS Note that the total Z range where the interpolation takes place can exceed the Z reference A and B 03 06 B 45 0019 e 4 85
52. resizing or movement of a mask element in the Image Display window Resize Click on handle Movement Click on line and hold the mouse button move the entire figure release mouse button and hold down the mouse button drag the handle release mouse button B 45 0019 e 4 255 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Closed polyline button Creation of a polyline figure in the image The first click sets the starting point each additional click adds a further line a click with the right mouse button closes the figure and ends the procedure Z Recycle bin button All the mask elements are deleted If one element was marked before this element is now deleted from the image Mask button Enables the Mask Mode where the region can be defined with ink Rectangle button Creation of a rectangle in the image Click and hold down the mouse button drag a rectangle in any direction release the mouse button to end the procedure 0 8 Ellipse button Creation of an ellipse in the image The first click sets the center point the displayed line permits the determination of the first dimension the second click sets the first dimension the second dimension and the rotation direction can then be determined the third click sets the second dimension and the direction and ends the procedure O Line button Determines the line thickness of the area outline Flood fill button Fills th
53. the entire program package image recording and analysis is available while only a part of the software 32 Bil NT Software functions image analysis only of already stored images Fig 4 2 Starting the LSM 5 software In the off line mode no hardware functions are available Of course the off line mode can also be started when the instrument system is connected In that case it is not necessary that the lasers and the microscope are switched on e Double click on the LSM 5 LIVE icon on the desktop of WINDOWS to start the LSM 5 software program Fig 4 2 The LSM 5 LIVE Switchboard window appears on the screen Fig 4 3 A Switchboard Carl Zeiss me LSm 5 LIVE Version 4 0 Scan New Images Ly Use Existing Images T Start Copyright Carl Zeiss 1996 2005 Exi Portions Copyright 1996 Microsoft Corporation All rights reserved xit Fig 4 3 LSM 5 LIVE Switchboard menu 03 06 B 45 0019 e 4 13 OPERATION LSM 5 LIVE Carl Zeiss Switching on the System LSM 5 LIVE DuoScan Online Mode Clicking on this button activates the complete LSM hardware on line mode RS Please note that the Online Mode button must be activated before clicking the Start button Otherwise the hardware can not be controlled by the LSM 5 software Offline Mode This item allows you to process and analyze previously acquired images ty with the LSM 5 software In this mode control of the hardware laser module is no
54. the following functions are not available VME button in the Macro Menu If your configuration does not include the Topography software package the following functions are not available Topo button in the Image Display window after acquisition of image stacks If your configuration does not include the Macro Recorder and Editor software package the following functions are not available New Save and Save as buttons in the Macro Control window Edit Step Delete Editor buttons in the Macro Control window 03 06 B 45 0019 e 4 327 OPERATION LSM 5 LIVE Carl Zeiss Software and Hardware Options LSM 5 LIVE DuoScan If your configuration does not include the 3D for LSM software package the following separate application is not available 3D for LSM If your configuration does not include the Multiple Time Series software package the following function is not available Macro Advanced Time Series If your configuration does not include the Image VisArt software package the following function Is not available 3D button in the Image Display window If your configuration does not include the Deconvolution software package the following functions are not available DCV Settings button in the Ortho function of the Image Display window DCV button in the Process menu If your configuration does not include the StitchArt plus software package the following function is not a
55. 0 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss The calibration results of the Basic Calibration will BasicCalibrationLog txt Notepad We of i be saved in a file named File Edit Format View Help BasicCalibrationLog txt to be found in the Current Objective Lens BIN Folder as well Epiplan Neofluar 20x 0 50 collimator calibration L Changes won t effect the stored values New Track Laser 488 Emission FilterLP 505 zoom colli 1 184 New Track Laser 405 Emission FilterBP 415 480 zoom colli 1 257 Current Objective Lens Epiplan Neofluar 20x 0 50 Pinnole Y Position Calibration New Track Laser 488 Emission FilterLP 505 Plate chLi Zoom Pinhole Y Position 1 458 New Track Laser 488 Emission FilterLP 505 NFT 488 chLi 7 oom Pinhole Position 1 459 New Track Laser 53 Emission FilterLP 550 NFT 532 chL1 zoom Pinhole Position 1 461 New Track Laser 635 Emission FilterLP 650 NFT 635 ChLi zoom Pinhole Y Position 1 457 New Track Laser 488 Emission FilterBP 495 525 Mirror Chl Zoom Pinhole Y Position 1 509 4 F F Fig 4 185 BasicCalibrationLog file 03 06 B 45 0019 e 4 185 OPERATION Macro Menu Carl Zeiss 4 8 6 Optimize Macro Optimize Channel 1 H Channel 2 o _ _ J Fig 4 186 Optimize window but can enhance image brightness efficiently 4 8 7 Relnit Macro Reset LIVE
56. 017 e gt 4 532mm 30 d F gt 639mm 01 gt u Fig 4 308 Scan Control window Channels cony confocal All e Click on the Channels button This displays the preset parameters of the contiguration loaded e Click on the Find button Make sure to position the slider correctly Then scan while the slider is in the LSM position This starts the scanning process The image is seen to build up gradually in a new window 3 Function Find produces images of different brightness for different scan speeds As a rule the first scanned image Pre Scan is not ideal since the photomultiplier is not matched to the light output More often than not the screen image is dull and needs subsequent optimization Ready 1024 1024 2 channels 8 bit Raw image data Display Zoom 1 2 Palette No Palette Fig 4 309 Image Display window 4 318 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 14 1 2 Confocal Aperture Detector Gain Ampl Offset e In the Scan Control window click on the Cont button see Fig 4 308 This starts a continuous scan e Use the Pinhole slider confocal aperture to set the aperture size in the Scan Control window under Channels The aperture size should be so small that there is still enough variation for the setting of the detector gain and that sufficient image information is still available 1 Airy is a good
57. 11 Select Crop This function allows to interactively define the size and orientation of a rectangular scan area on the image displayed in the Image Display window The defined area is displayed together with the Zoom and Offset parameters in the Scan Control window in the Mode Submenu Click on Crop will display the Crop Rectangle in the Image Display window Any changes done with the Crop Rectangle are setting the parameters immediately On the next execution of eee a scan Find Fast xy Single Continuous in Fig 4 252 Image Display window Select Crop Scan Control or Start T or Start B in Time Series Control these new scan parameters will be used Feri S92 oo S02 2 Johara phi To reset the crop function and use default values set Zoom 1 and Offset 0 in the Scan Control window in the Mode submenu e Click on the Crop button The Crop Rectangle will appear on the Image Display window The Crop Rectangle is controlled via the following functional elements Offset a e Click into the crop rectangle keep the left mouse button pressed and drag the crop rectangle to the required position Release the mouse button Zoom 7 e Click on a corner of the crop rectangle keep the left mouse button pressed and set _ the required size Release the mouse button Side ratio 4 Click on any of the Intersection points between crossline and crop rectangle keep the left mouse button pressed and chang
58. 2 D Zmin Z Zmingt 111 2 25 Averaging of R values of all the 25 single area elements When combined both parameters provide information about the homogeneity of the surface Big differences are indicative of pronounced inclination of the overall area or of spikes maxl min max2 max 25 Z min25 maximum roughness depth RS m x RS aax Max z Z Z maxl min1 max2 mina Zmax25 EZ ne maximum of R values of all the 25 single area elements iL Both the roughness parameters and the z histogram will be influenced by the use of filters 03 06 B 45 0019 e 4 303 OPERATION Carl Zeiss Display and Analysis of Images 4 13 24 8 3D Measurement Functions 1 Volume measurement mode Flood function e Use the 3D button to select the required 3D display of the stack e Click on the Volume button in the Measure button bar The volume parameters are calculated and displayed below the image LSM 5 LIVE LSM 5 LIVE DuoScan The Copy button is displayed below the right hand side of the image This button permits the volume values to be copied to the clipboard and imported to other programs e g MS Word or MS Excel via the Paste function e Setting the Fill Level slider enables you to change the height level of the topography The portion of the topography lying below the set height level is filled with water blue color and the volume parameters are calculated online only for the pro
59. 214 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan e Click on the Close button to exit the Objective Control window and accept the settings 4 10 3 Pinhole Adjustment In the Pinhole and Collimator window the aperture are optimally aligned and adjusted to the used beam path configuration The position of the aperture pixel shift and Y coordinate in relation to the detector makes a major contribution to image optimization In all existing standard configurations the apertures have already been adjusted at the factory These settings are taken over for active operation when a standard configuration is loaded If you want to create a setting that differs trom the standard configurations adjust the aperture as follows LB The X position does not influence brightness but shifts the whole image position in X direction L Use the Optimize and Kollimatic functions see chapter Macro Menu first the Pinhole Adjustment is not recommended on the LSM 5 LIVE as a general tool Fig 4 226 Fig 4 227 OPERATION Maintain Menu Carl Zeiss TS Objective Control xj Partocal Correction Activation When parfocal correction i active the moving Notize stage can dammage your objective i Parfocal corection active Parfocal Correction Adjustment Epiplan 1008 0 75 Epiplan 1008 0 75 Curent Objective Nest Objective Objective Epiplan 1008 0 75 will move to the light path Get in focus with
60. 2D display mode 4 306 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 Curve of tp measurement mode in 3D display This function is performed in the same way as in the 2D display mode Before determination of the tp bearing portion individual peaks noise steep slopes must be eliminated The Median filter or a 3x3 longpass filter can be used for this purpose Pac URE pr Ful OF 3 Arey O44 S Meade 217 0 Sie 1S charai i Fig 4 300 Image Display window Topography display 3D Curve of tp Shifting the two cursor crosses permits two bearing portions to be given in percent e g Smr1 10 Smr2 90 as default values for which the height difference Rdc is determined automatically 5 Grad Histo measurement mode in 3D display This function is performed in the same way as in the 2D display mode 6 Roughness measurement mode in 3D display This function is performed in the same way as in the 2D display mode 03 06 B 45 0019 e 4 307 OPERATION Carl Zeiss Display and Analysis of Images 4 13 24 9 Export Data multiple profiles Rel 3 2 single profile parameters topography as matrix topography as triples 4 13 24 10 Topo ReUse Topo routines can be saved and reloaded as tgp files TopoGraphic Parameters These files include settings for reconstruction mode intensity threshold filters including FFT tilt angles manual 3 poin
61. 3 4 7 2 4 4 7 3 4 7 3 1 4 7 3 2 4 7 3 3 4 7 3 4 4 7 4 4 7 4 1 4 7 4 2 4 7 4 3 4 8 4 8 1 4 8 2 4 8 2 1 4 8 2 2 4 8 2 3 4 8 2 4 4 8 3 4 8 4 4 8 4 1 4 8 4 2 4 8 4 3 4 8 5 4 4 OPERATION LSM 5 LIVE Purpose LSM 5 LIVE DuoScan Single Wavelength Dyes Offline Calibration sessssninuesssrinnssrrrirsnrrrrrrerrrrrenen gt 4 142 Ratiometric Dyes ssnussinssinesinesiurrnrittrrtt cee 4 143 Ratiometric Dyes Online Ral Oi ecccinsevocennel aivsi seweteedueueronabaanatnyenied calles Aamalesanotasentien 4 143 Rauometie Dyes Gal ail Ol irasra a T A E aT 4 144 Ratiometric Dyes Equation Calibration Grynkiewicz s sesieessieerieerrrrerrrerrrreeen 4 145 Options for Calibration Image Selection Equation or Titration Calibration 4 145 HNIC SC NNN E E E P A gaan E T EE E er toareener ainda ence berets 4 145 COO VIM OW CGN CIS e E EE EA E EEE 4 145 CODY FUR OUO eesi e E E E EE E 4 146 Paste BItMAap sciaaiscaceotmntntnndncedinngeatcnantunndtatanctnepetedeondieantdvenaneieeuiedclndeniadnernendndsaaaaiesanetind 4 146 ING EEEE E EAE O TE A E T E ETE TEA T E EENE 4 146 Open Close the Kinetic Analysis WINGOW ss sssiiusssiinssriruesrrressrrriesrrrrerrrrrerrrrrenrn 4 146 FUNCION DECIDU O ersen E EAEE A E E 4 147 Example FRAP Performed in a Nucleus Expressing GFP Labeled Proteins 4 148 SD NVICW MENU eer ne eee ee eee eee eee eee eee eee eee 4 152 3D DepthCod Colo
62. 4 5 6 1 4 5 6 2 4 5 6 3 4 5 6 4 4 5 6 5 4 5 6 6 4 5 6 7 4 5 0 8 4 5 7 4 5 8 4 5 8 1 4 5 8 2 4 5 8 3 4 5 8 4 4 5 8 5 4 5 9 4 5 9 1 4 5 9 2 4 2 OPERATION Purpose E ECONO a E E E A A Switching on the Enterprise UV Laser cccccccecccceeeceeeeeeeeeee scenes Opening Closing the Laser Control WINdOW c0ccceeeeeeeees FUMCHOMWESCHIOTIOMN snncuxeswesanrctetatatdenatentsaukeuneetakacechmiatantunaaieatss PUTING waracerecntachonceueteuindnantndestarinsaunedinchduntentating tiaiaierteancteaameaciccens Microscope COI ON aa sceisrsaeectenrouaneesrntnrascetadgantemadanaatedenantedees aetyynteiuauiand Open the Microscope Control WINdOW 0cccceceecceseeeeeeeeeeeees Microscope Control Window for Axio Imager Z1 o s Microscope Control Window for Axiovert 200 M secen Working Im LSM MOU Geseri anr er ee nee ener eee Microscope Control for Axioskop 2 FS MOT COP Ua OM COIS Oleri ese n EEE EE Open Close the Configuration Control Window 0 SOC CU BUON rentes on nederisa eeraentngrnsencantahiaetaenieieieunwneneenimematiaceneins OS DENO attest ote E aceueh cataara eccteos a cetece ates Settings for Single Track in the Channel Mode scce Settings for Multi Track in the Channel Mode seeen Transmitted WON ORCS ac deetendaerantadnueoreu eee eeienssranbensceteouerconserees Ratio Settings Panel ccceccccececcccce see eeeeeeeeeeeeeseeeeeeeeeeeeeeeeae es Camera Detection Panel 0 ccc
63. 4 beads for each track are found within the green rectangle Jse Arrow keys or DropDown list to change the rak You mayi use Laser Power Integration Time Exdude Current Track Laser Power d ja Integration Time J i i Current Collimator LIVE_Coll_RGB J e Fig 4 176 Figure Sample and Configuration Setup Window of the KolliMatic Macro 4 179 LSM 5 LIVE OPERATION Macro Menu LSM 5 LIVE DuoScan Carl Zeiss f th HDF Sih Mii viveyt Wa iij r fpa i nil he W KI lyy a i ONN V IK i aul K it M ie yada a a Finely SIZESIS 1 ghara H bel Fig 4 177 Beads focused for maximal vertical thinness imap T5 AIM aee MPA E a a 5 lal Fig 4 178 Beads optimally focused B 45 0019 e 4 180 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss Running the Basic Calibration ms If you have selected the check box for Basic Calibration please follow these instructions In order to run the Calibration Matrix only move on to paragraph 7 e Make sure that both check boxes have been m eA A AAA selected Fig 4 179 E io a P e Double check if the objective settings shown at TRE E Tri pf the bottom of the macro window match the aati currently used objective a TON am e Click the button Calibrate e In order to follow the calibration process just click into the Message window a scroll bar will appear e T
64. 45 0019 e 4 329 OPERATION LSM 5 LIVE Carl Zeiss Courses on How to Operate the System in an Optimized Way LSM 5 LIVE DuoScan 4 17 Courses on How to Operate the System in an Optimized Way Carl Zeiss is offering training courses on how to operate the system in an optimized way Courses are held in our application center in Jena Germany Courses are held in English or German language respectively Check out www zeiss de Ism for the latest dates and ask your Zeiss representative for a quotation on courses 4 330 B 45 0019 e 03 06 LSM 5 LIVE TOOLS LSM 5 LIVE DuoScan Contents Carl Zeiss CHAPTER 5 TOOLS CONTENTS Page 5 TOOLS esi a aun eecepaeemosaecndetee EE aouueeeceaeeeeaes 5 2 Del Vie CO RETS apaa a E tee ake eee sapere ee ia aac 5 2 5 2 SA CSC leases esta a ada I AEE TE E EEEN E ENE 5 4 9 3 CONEIS GE BLONS E rrna E aorta teeter maten eet din 5 5 5 4 EES reer aN lr ot cram kcegeecton da te capcutematile ciate coeeece latent aie see cca nateeneseceanea nace eee as 5 6 03 06 B 45 0019 e 5 1 TOOLS LSM 5 LIVE Carl Zeiss Change Filters LSM 5 LIVE Duo Scan 5 TOOLS 5 1 Change Filters The Change Filters tool is used to update the filter data in the software after a change of filters in the reflector turret of the microscope the emission filters and the beam splitters of the LSM 5 LIVE All filter data are updated in the Emission Filters amp Beam Splitter Control window After activation of the appropriate button
65. 45 0019 e 6 21 Carl Zeiss 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan The Output histogram shows the resulting histogram They are not editable The horizontal axis represents the grey values from O to the maximum which is either 255 or 4095 depending whether the input is 8 bit or 12 bit The vertical axis represents the pixel count The vertical scale of the histogram is set using the scroll bar The units are percentages of the grey value distribution maximum This setting has no influence on the function Parameters Input Output Threshold Input L Input C Input H Output L Output C Output H 6 22 Input image sequence Output image sequence Exclusion value 0 1000 Lower boundary of grey value range Input Center of grey value range Input Upper boundary of grey value range Input Lower boundary of grey value range Output Center of grey value range Output Upper boundary of grey value range Output B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Contrast Linearize This function scales a range of grey values of an image sequence to equal area fractions in the histogram 4 Contrast Ea Interactive Automatic Linearze Input ft ma Output Booo Hew Channel Alf 1 Skip Black m Input Histograrn utput Histogram Fig 6 15 The Linearize tab sheet of the Contrast dialog window must be selected This function enhances the contrast by li
66. 488 nm Check box Activates deactivates setting options Button Close l l l Selection performance of a function via mouse click 4 10 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Purpose Carl Zeiss 4 1 3 Convention for the Text in this Manual All the originally used terms of the software interface e g names of windows panels input boxes list selection boxes check boxes menu items names of buttons and keyboard keys are displayed in bold letters to allow easier identification 4 1 4 Backup System backup A complete backup is contained on the enclosed backup CD ROM User files backup The following user generated files need to be included in a backup procedure keep directory structure Image database files mdb but not system_configuration_ mdb LSM Image files lsm Exported images Tiff LSM Tiff BMP Palette files AIM Palette lut Filter files AIM Filter krn Pibhole aperture setting files AIM PH pos Log files AIM log The following files generated during the system integration should also be included in a backup procedure Parameter file for pinhole aperture setting AIM set Parameter file after pinhole aperture adjustment AIM ad Scanner Tiles AIM bin bin Microscope stand database AIM database system_configuration_ mdb 4 1 5 Software Operati
67. 6 2 6 1 OMETVICWY TEXDAN ON aaa a e E A E 6 2 6 1 1 TASIM GES CGU EN E n E a E EE EA 6 2 6 1 2 TREMIJE PRO Denes e e e r a a Ea S 6 3 6 1 5 KOROV cys 6 erga mre A ee ere rete eae tener one ee ae ne nee ee eee ene 6 3 6 2 OEE ala E E E RE E E EE EAN ee re 6 3 6 2 1 iair oe 1a cea a EE EE ES en me oe AEAEE N EE A S EAA EEA eT AEEA 6 3 6 2 2 WAA TE EN EEE EE EEE Coe EEEN AEE ETETE E N E NE A ENE 6 4 6 2 3 Pispa VV NIN COW enri Seleagcuan aid dese dohaicanaieyctinetbaghan aunt ken Oolouahtstantbnhatenueienndeneectanes 6 7 6 3 FUNC BIC IAS enerne a a astra aa a a a poe ees tana sade Bong ones a a hones 6 11 6 3 1 Functions in the File MenU cccceccccecececececececcecececececececeeececeuauaesesesesecssscecevereeeenenenes 6 11 6 3 2 Functions in the Edit WGN Ulis 3 actw sederie ne san eenborci eea oboe and alea eAtee hansen ae 6 14 6 3 3 Functions in the Process MenU cccccccececececececcccccesccecececececcececeaececsuecsadeueueauaeneseueeesecs 6 17 6 3 4 Functions in the View MenU cccccccecececececececcccecececececececeseceecuausesesecececseutaueeauaenersness 6 43 6 3 5 Functions in the Measurement MenU 2 0 ccccccececcecccccccccecececcucucecucacacececeueusueneucuseceeeeeenens 6 50 03 06 B 45 0019 e 6 1 3D FOR LSM LSM 5 LIVE Carl Zeiss Overview and Explanations LSM 5 LIVE DuoScan 6 3D FOR LSM 6 1 Overview and Explanations 0 0 0 Voxel 6 1 1 The Image Sequence The 3D for LSM handles image sequences
68. Binary W Invert i Input Histograrn L li 4p E 127 pl H 255 WHO 255 0K Cancel Fig 6 25 The Automatic tab sheet of the Segment dialog window must be selected Segmentation is especially used to generate binary regions These are required for the measurement The function calculates the two strongest local minimums in the histogram of the Input image sequence These values are used for the discrimination Only one channel of a multichannel sequence can be selected as Input Output will always be a single channel sequence The vertical scaling of the histogram can be adjusted with the scroll bar at the right edge of the histogram This setting has no influence on the function The Green and Blue Red option buttons of the parameter Colour determine whether the voxels within Green or outside Blue Red of the grey value interval L H are displayed with the corresponding colour If Green is selected the voxels within the selected interval are highlighted in green The rest of the image retains its original grey values The voxels with the grey values Low and Low 1 are displayed in blue The voxels with the grey values High and High 1 are displayed in red If Blue Red is selected the voxels with grey values within the interval Low High remain unchanged Voxels with grey values less than Low are highlighted in blue those with grey values higher than High are highlighted in red If the Invert option is selected the grey va
69. Close button to quit the window 4 6 9 2 Image Panel The image to be processed is selected in the Image panel The currently selected image is shown in the display box of the image selection box Proceed as follows to select a series via the image selection box OPERATION Process Menu Carl Zeiss 4 Interpolate Brightness and Contrast Interpolation Start Image Brightness Contrast g End Image Brightness Contrast Ch 1 M Overwrite Source Images I Ignore Images less than Start Image and greater than End Image Fig 4 124 Interpolate Brightness and Contrast window Click into window Convallaria Stack Fig 4 125 Image panel e Click on the arrow button The image selection box will be opened and all the currently loaded images will be displayed in a minimized form e Click on the required image which will then appear in the display box of the image selection box and has been selected for the interpolation procedure 3 You can also use the Click into window button for image selection 03 06 B 45 0019 e 4 133 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan nterpolation 4 6 9 3 Interpolation Panel Start Image i ll First Fh In the Interpolation panel the parameters for the HeU aa ee al el interpolation procedure are set n Z Contrast d M e Use the Start Image slider to select the slice at g which the interpolation
70. Convallana Display Hy GE E Split sy Ready 512 x 512 x 20 hannels 3 bit Overlaid image Fig 4 253 Image Display window Split xy display 3 This function is useful to optimize the individual channels in a multi channel image acquisition together with the Range Indicator palette 03 06 B 45 0019 e 4 245 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 17 Display Ortho This function allows to display a Z Stack of images in an orthogonal view measure distances in three dimensions generate 2D deconvolution views of the yz and xz plane The settings of Chan Zoom Slice Overlay Contr and Palette apply Click on Ortho will be immediately effective e By clicking on the Ortho button section lines appear in the Display toolbar together with orthogonal projections in the image On the right hand side the Orthogonal Sections toolbar is shown convallasia AIM ol i i Select Display Orthogonal Sections YZ plane red AZ Plane green Section result YZ plane Ready 512x512 x66 3ct k bil Section result ale Fig 4 254 Image Display window Ortho display 4 246 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 17 1 Ortho Select Function By changing the parameters X Y and Z in the Orthogonal Sections toolbar the section plane can be pos
71. Display window e Click on the arrow button of the image selection box to open this box e Click on the relevant image if the Add operation shall be performed in an existing Image Display window or e Click on New Image 8 bit or New Image 12 bit to use a new Image Display window 3 Youcan also use the Click into window button for image selection Clicking on the New 8 bit or New 12 Bit button enables you to determine directly and quickly whether the new image is to be created in the 8 bit or 12 bit format 4 122 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss If an existing Image Display window is used to perform the Add function you must determine whether an existing channel shall be overwritten with the Add operation or whether a new channel shall be added e In the Channel selection box click on the channel which shall be overwritten or click on New for a new channel 4 6 1 4 Add Panel Source 1 14 00 Source s 4 00 In the Add panel the currently set formula for the L o Ldn Add operation is displayed The editable input c boxes permit the formula to be changed with any numeric values Fig 4 110 Add panel e Click in the required input box and enter the relevant value e Click on the Apply button to perform the operation in the activated window or a new Image Display window e The new image can then be stored via the Save As function 4 6 1 5 Preview Panel W Pr
72. DuoScan Display and Analysis of Images Carl Zeiss 6 Show processing parameters After selection of the Show processing parameters function a reporting of the following applied topo processing functionality is displayed on the right hand side of the Image Display window Mode calculation mode Max Center First Threshold applied intensity threshold Filter Fit plane cylinder sohere parameters Com al Ferd Tiza S12 15 Tehran B bel Fig 4 289 Show processing parameters 7 Ratio of valid data points The ratio of valid data points means signal intensities within a given intensity threshold is displayed 8 Load Calculation Parameters Topo routines can be loaded as tgp files TopoGraphic Parameters 9 Save Calculation Parameters Topo routines can be saved and reloaded as tgp files These files include settings for reconstruction mode intensity threshold filters including FFT tilt angles manual 3 point fit fit procedures plane cylinder sphere inverse fill holes 03 06 B 45 0019 e 4 289 OPERATION Display and Analysis of Images Carl Zeiss Render properties Renderer Fig 4 290 Context menu of the 3D display mode Shaded 3D Rendering E Shading Model List Default Fig 4 291 3D Rendering window LSM 5 LIVE LSM 5 LIVE DuoScan 10 Render properties item This option is only available in the Shaded 3D display mode Use this fu
73. Emission Filters Filter Cubes Stand or Beam Splitters LSM the special input mask for the selected filter type is displayed Since the procedures of updating or entering a new filter type are identical for all types only the updating of filters in the reflector turret Filter Cubes Stand will be described in the following e Close the LSM 5 LIVE software program e Insert the new filter module in the reflector turret e Double click on the Change Filters icon on the desktop The Emission Filter amp Beam Splitter Control window appears on the screen The name of the currently used database is displayed in the System Database box with the filter type being indicated below for checking purposes e Click on the Filter Cubes Stand button The Filter Cubes Stand panel is displayed The Filter Cubes Stand panel shows the Filter Wheel No and the filter positions available Use the Name and ID selection boxes to enter the filters installed in the individual positions of the filter wheel e Open the Name or ID selection box of the relevant filter position and select the new filter set from the list e Click on the Store button to accept the new settings e Click on the Close button to close the Emission Filter amp Beam Splitter Control window L All available filter sets have to registered in the filter list see Edit Filter List function next page Edit Filter Beam Splitter List x Edit Filter List EB Close The Edit F
74. Font button enables you to select the font style and size in the Font window The entered text will be displayed in the left upper corner of the Image Display window after clicking on OK and can be moved to the required position using the mouse The Text window can also be activated with a double click on a created text box and the entered text can be edited subsequently Insert opens up a further window which allows you to annotate coordinates time and Z position with either automatic or user definable units and precision This annotation is updated during image acquisition and can be exported with the image The annotation can be stamped into already existing images 03 06 B 45 0019 e 4 35 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan Recycle bin button All the overlay elements and dimensions dragged to the scanned image are deleted If one overlay element was marked before this element is now deleted from the scanned image Line button This button allows you to determine the line thickness of the area outline fa Color button After clicking the Color button the Color selection box will be opened The colors displayed in the Color selection box can be assigned to the overlay elements with a click of the mouse The currently selected color is displayed in the Color button A selected color is automatically assigned to the currently selected overlay element and then to all the elements created afterw
75. LSM 5 LIVE Carl Zeiss Specification of Trigger Interface LSM 5 LIVE LSM 5 LIVE DuoScan 7 7 Specifications of Trigger Interface LSM 5 LIVE Application With the LSM 5 LIVE you can control various actions externally using Trigger In or force external devices to work at a defined time depending on an action using Trigger Out during time series These actions are Scan Start Stop Bleach Change of Scan Interval end of a countdown set marker into image or even a mouse click on a button Interface User Port Adapter on the back of the Real Time Control Unit in the ECU of LSM 5 LIVE Connector 2x high density D Type plug 1x coax with outer shield Number 8x signal IN all able to generate an interrupt 18x signal OUT 10 synchronous 8 asynchronous relative scan position 1x clock Connector Coax Triax Lemosa Serie 00 EPL OO 650NLN Pin Signal name Description Signal PCLK_Out PixelClock Output Outer shield Shield Chassis ground 7 10 B 45 0019 e 03 06 LSM 5 LIVE ANNEX LSM 5 LIVE DuoScan Specification of Trigger Interface LSM 5 LIVE Carl Zeiss High density D Type 15pol Plug A Pin Signal name Description SyncOUTO Synchronous output e mon oosa ooo e pomon osoa Ooo o wmon osoa ooo High density D Type 15pol Plug B Pin Signal name Description e omon oosa O ooo e mons oosa ooo o mon oso oo pull to signal ground depending on internal switch inside rea
76. Method PsF The Deconvolution panel contains the two tabs menog Inverse Method and PSF Restoration Effect weak Strong Auto detect 1 Method tab The Method tab permits selection between the calculation methods Nearest Neighbour Inverse and Iterative a Nearest Neighbor Fig 4 166 Method tab The Nearest Neighbor method is the simplest and fastest algorithm which in principle corresponds to a 3D sharpness filter b Inverse Filter The regularized inverse filter generally achieves better results than the Nearest Neighbor algorithm It is well suited to process several image stacks for a pre selection of images for the use of the iterative high end methods c Constrained Iterative The best image quality is achieved using the Constrained Iterative Maximum Likelihood Algorithm Increasing the resolution in the image especially in the Z direction is only possible with this method Due to the complex mathematical method depending on the image size and the PC being used the calculation can take up to several hours In the Inverse method the Restoration Effect slider permits the noise to signal ratio to be selected between the settings Weak low noise and Strong pronounced noise 03 06 B 45 0019 e 4 165 OPERATION LSM 5 LIVE Carl Zeiss 3D View Menu LSM 5 LIVE DuoScan Activation of the Auto detect check box will start a routine for the automatic determination of the noise level in the entire image part o
77. Number of C Blue A Reset al Fig 4 250 Color Palette window 4 238 4 13 8 1 Editing and Storing a Palette A palette is edited by moving the knots in the Ramp Polyline and Spline functions identical to the setting in the Contrast and Brightness window see page 4 234f The palette can be set for all colors together or separately for each color e Activate the relevant button Red Green Blue or All Proceed as follows to store an edited palette under a new name e Click on the Add To List button the Add Palette To List window will be displayed e Enter a name for the palette and click on Ok The palette will be stored and the name included in the Color Palette List panel B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 8 2 Delete a Palette Proceed as follows to delete a palette e Click on the name of the palette to be deleted in the Color Palette List panel and then on the Remove button The palette will be removed trom the list 3 The standard settings No Palette Range Indicator Glow Scale and Rainbow cannot be deleted 4 13 8 3 Import a Palette Proceed as follows to import a palette e Click on the Import button The Import Palette window will be opened e Select the required palette Tile extension lut from the relevant directory and click on Open The palette will be imported and displayed in the Color Palet
78. OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 9 4 Tile Scan Panel Tile Numbers p x p y S This function permits a frame to be created as an Mark overview image of the specimen with a maximum size of 4096 x 4096 pixels According to settings such a frame is divided in XY tiles of 1x 1 to the maximum of 15x15 A tile of special interest target can then be selected for scanning Tile Size x 512 Pixel Y O12 Pixel Frame Size s 5 2 Pixel Y 512 Pixel 927 268 um f 921 28 um Fig 4 104 Tile Scan window The application of the Tile Scan function requires an objective with a minimum magnification factor of 2 5x Tiles Numbers X Y input box Input of the number of tiles for X or Y from which the frame is to be composed Tile Size X Y display Display of the size of a single tile in um corresponds to the value selected in the Scan Control window Frame Size X Y display Display of the frame size of the tile scan for X or Y Specification in pixels and um Move To button If the Move To button is activated a rectangle with a target allowing the selection of the region of interest is positioned in the center of the scanned frame Click and hold down the left mouse button to drag the rectangle to the required specimen area When you release the mouse button the stage moves to the selected position Mark button If the Mark button is activated marks previously set in the Tile Scan image ar
79. PMT to create an image with the DuoScan system e Start the Image Match Scanfield Transformation macro e Use the Image match calibration sample delivered with the system This sample has a fluorescent layer and a reflective grid useful to create images in fluorescent reflective or transmission light mode e Start continuous scan and set laser power and gain to appropriate levels The point scanning system will be the speed limiting system so choose a fast scanning speed to get a faster image refresh e Use unidirectional scan to avoid adjustment errors due to bidirectional image jitters e Set the matching zoom factors with one of the Match DuoScan zoom offset or Match LIVE zoom offset buttons relative to each other It is recommended to adjust the LSM zoom Z LSM 5 LIVE File Acquire Macro Window Process 3D View Options Maintain Help i Acquire Process ae aD View x AA ra a J N l _ _ Macro Visual C ism DuoScan OrFsek x OFFset Y Amplitude Fotation Test grid E Match DuoScan zoom offset Match LIVE zoomforFset Track CHO i Track ChLI Store pply Single Track Contig Add Track Remove Fig 4 188 Scanfield Transform window These buttons set the two scanheads at a matching scantield diameter according to the following table LSM 5 LIVE Zoom 1 0 Microscope stand LSM 5 LIVE DuoScan L
80. Pixel Depth Scan Direction amp Scan Average Data Depth amp Bit 12 Bit Mode Frame Method Mean Scan Direction rm aber C ai Single Note When using an Axioskop 2 FS MOT indicate the objective that is in use in the Scan Control window This ensures correct calculation of pinhole Z stack optimization etc Stop Adjusting the exposure time e Use the Exposure Time slider in the Objective Fig 15 Scan Control window Mode settings Lens Image Size amp Line Step Factor panel Fig 15 to adjust the exposure time Below the slider is shown the number Frames per Second Start with 2 to 4 frames per second Choosing the dynamic range e Select the dynamic range 8 or 12 Bit per pixel in the Pixel Depth Scan Direction amp Scan Average panel Fig 15 8 Bit will give 256 gray levels 12 Bit will give 4096 levels Publication quality images should be acquired using 12 Bit data depth 12 Bit is also recommended when doing quantitative measurements or when imaging low fluorescence intensities Setting the scan averaging Averaging improves the image by increasing the signal noise ratio It can be achieved line by line or frame by frame Frame averaging helps reduce photobleaching but does not give quite such a smooth image e Select the Line or Frame mode for averaging e Select the desired scan average method Mean or Sum in the Method selection box If you are using the Mean method the
81. Selecting the Cancel button does not execute the function restores the parameters and closes the dialog window Pressing the Apply button executes the function with the defined parameters the dialog window will stay opened 6 2 2 Main Window The Main window includes the Menu File Edt Process View Measure Windows Help the Tool bar label obet 3 6 4 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan File Menu Open Image Save Image As Save Display As Print Exit Edit Menu Copy Edit Channels Delete All Images Process Menu eae Arithmetics Contrast de Smooth one bas Morphology H Segment E A Boolean 03 06 3D FOR LSM User Interface Carl Zeiss Opens a Tile selector dialog to load an image sequence Opens a Tile selector to save an image or image sequence Saves the currently shown contents of the Display window as a single colour image The printer parameters can be set with this tool The standard Windows printer dialog is opened Terminates the application Copies the contents of the Display window to the clipboard Allows to add or to remove channels to a single or multichannel image Deletes all images and image sequences trom the memory Adds or subtracts the grey values of two image sequences Add Subtract Enhances the contrast and brightness of an image sequence Interactive Automatic Linearize Smoothes an image sequence P
82. Settings Stack Z Size The dimension of the Z Stack in um The stage nosepiece is moved in such a way that the stack size dependent on the refractive index is achieved optically Focus Position The current Z position If the refractive index Refr Corr changes the value of the focus position in relation to the 0O also changes online Z Slice Opens the Optical Slice window Fig 4 63 The Optical Slice window contains three buttons Optimal Interval um Optimal Pinhole Diameter confocal aperture and Undo to allow the setting of the optimal interval and the optimal aperture size of fluorescence stacks Both values influence each other and depend on the objective used In the case of a fixed aperture size half the value of the smallest aperture size used Is taken to determine the optimum interval Accordingly the aperture size to be used in the case of a preset interval is determined by doubling the value of the selected interval Undo resets the just before altered values The Optical Slice window displays the following information Black Stack Z Size um intervals x number of slices 1 Optimal Interval depending on the objective used and the aperture size setting Red and other colors Presentation of the actual data set by the operator helps to optimize stack creation 03 06 B 45 0019 e 4 79 Carl Zeiss OPERATION LSM 5 LIVE Acquire Menu LSM 5 LIVE DuoScan TS Optical Slice kiti Stack Z Size 19 64
83. Size IV Wavelength V Free space on local harddisks Vv Scaling lV Position MV Memory amount for next scan IV Average V Image memory type Fig 4 207 Scan Information tab Peer Import Export Scan Information Print Status Display Sevessnessnsnsesssesesesssssssssssesssesssssssssssssssesssssssan Show status diplay upon opening of a new image display m Status display in image window Image statusbar MV Scan Mode MV User V Stack Size Pixel MV Scaling Detector Gain MV Number Channels V Stack Size um Amplifier Gain Raw Reconstruction V Zoom Amplifier Offset V Pixel Depth MV Objective V Pinhole Pixel Intensity Mouse T Pixel Time IV Filters l Display Zoom I Position V Wavelength Palette V Average MV Beam Splitters Fig 4 208 Image Status Display tab Image Status Display T Import Export Scan Information IV Print Status Information M Scan Mode M Position Iv Filters M Scaling Average Vv Wavelength M Stack Size um MV Detector Gain M Beam Splitters M Zoom M Amplifier Gain MV Processing Summary M Objective lV Amplifier Offset User MM Pixel Time M Pinhole Fig 4 209 Print Status Display tab 4 9 2 7 Scan Information This tab permits the setting of which scan information shall be displayed in the Scan Information window see Window pulldown menu of the Main menu page 4 222f Activation deactivation of the information to be display
84. Under Scan Number you must enter after how many scanning procedures bleaching is to be performed Number of Scans in a time series after performance of which the bleaching procedure shall be started If this check box is ticked b the bleaching procedure is automatically performed in combination with a time series Under Scan Number you must enter after how many scanning procedures bleaching is to be repeated Number of Scans in a time series after performance of which the bleaching procedure shall be repeated If this check box is ticked I you can set the current stage position as the one in which the bleaching will be done by clicking the Mark Position Z button This function is only available using the Line or Frame scanning mode If this check box is ticked J the speed for scanning during the bleach process can be defined independently of the scan speed during image acquisition A lower speed results in a longer pixel dwell time which increases the efficiency of bleaching If this check box is ticked J you can set a different XY position for spot bleaching This function is only available using the Spot scanning mode Click on the Spot Select button in the Scan Control window A new image is produced and two crosshairs appear in the image The red crosshair marks the spot that will be imaged The green crosshair marks the spot that will be bleached Move the center of the crosshairs to the desired positions and perform bleaching
85. View button Shadow rendering with user defined view 4 268 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan Transparency projection Basic button Advanced button Surface projection Basic button Advanced button Full Resolution button Appearance button Series button OPERATION Display and Analysis of Images Carl Zeiss Transparency rendering voxel based Transparency rendering voxel based with textures Surface rendering voxel based Surface rendering triangle based High accurate surface rendering triangle based Opens the render properties dialog Renders a series of 3D image stack or 3D 4D time series opens the Series render dialog 4 13 23 1 Shadow Projection With a click on Front the 3D reconstructed image is displayed in a shadow projection where it is illuminated at a 45 angle from the front left A click on the Back button creates the same projection with illumination from back left f Convallaria AIM l0 x Display Fes Any View Transparency A Basic Advanced Surface Basic Advanced Full Res Appearance Series Ready 512 x 512 x 20 3 channels 8 bit Fig 4 272 03 06 Image Display window 3D display Shadow projection Front view B 45 0019 e 4 269 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan The zoom wheel to the left of the Image Display window allows contin
86. WIC OM aa cpecnsceetorenaasaaboricouatwakoib AA E sansa ee stencuanks 4 121 SONCE Elea easeger sentient a hie ceast Net saan fn ce ia ocmeslies st Cueahs 4 122 Destina on ets 2 Peper nee nate rier een a geen ee ate Rte nO Yate nt enn eon ee nee re ea ean 4 122 PGI EP Cassone E A A oie tnt Goeeae bas cues uate aoa acs E N 4 123 Fe NSM Me AI EEEE E E aceuieau AE N E E E E AE EAE 4 123 Sl E EEE EA AETS E E EE A E A AET 4 124 Opens Close the SUBT dCEWINGOW ranea e ee a aT A 4 124 Pertormance ot the Subtract FUNCION misrioieeicutseeinrineraen n i EIE 4 124 PUO aa a a e a a a a E E A E 4 125 Open 7 Close the M ltip y WInNdOW eetere en e aaka 4 125 Penornance ot the Muhip y FUNCOM ra Nites ae clit atts eter ana ciel ated tes 4 125 REI Ree etc eter eine eT ra eo eee 4 126 ODEN Close The Ratio VVINGOW aici naa e E ddan dnaeecdabindi eesaeenee 4 126 PEROLMANCe OL the RAO FUNCH ON pes vacheradecectecannaeacts ancient cestengneetenteusoatncgutadse e atacssaeae 4 126 FG Fe Meteo sects Sessa eee Sige aacacs aetna E EA ace E a annie N 4 127 Ge oN s CS alc alge apres aren eee ene ic ee ere cn Tenn ener ene ee TE eee oe ee 4 127 open Cose Ine GOD VIN GOW cerita alee none E AE teal ae 4 127 PEON MAnCe Or the Copy FUNCION eenn cout targa alee den bce ade eat ot elena 4 128 Pupe Onm aCe sssansc eA AR E E EN Seema oeastotan 4 128 PTE Me EE EA AIEA TE A E TE AE A E A A AEE 4 129 Open A close the Filter WNdoW ameona E a Gaia 4 129 A PAN ea A E EE O A EI 4 129 Pi
87. Z Stack must meet the following requirements At least two fold oversampling in xyz z halt of optimal interval button High signal to noise ratio Detector gain lt 500 V Calculation is either made for one channel of the opened image which must first be selected accordingly or for all channels of a stack Calculation is started via Apply and can be stopped using the ESC key if required 4 7 4 1 Open Close the 3D Deconvolution Window e Click on the DCV button in the Process subordinate toolbar of the Main menu This opens the 3D Deconvolution window e Click on the Close button to quit the window 4 164 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan 3D View Menu Carl Zeiss 4 7 4 2 Source Panel Click into window Channel The image to be processed is selected in the Source panel The currently selected image is af i shown in the display box of the image selection Convallaria cni Ef box Proceed as follows to select a image via the image selection box Fig 4 165 Source panel e Click on the arrow button The image selection box will be opened and all the currently loaded images will be displayed in a minimized form e Click on the required image which will then appear in the display box of the image selection box and has been selected for the interpolation procedure IB You can also use the Click into window button for image selection 4 7 4 3 Deconvolution Panel
88. Zeiss User Interface LSM 5 LIVE DuoScan 1 14 User Interface Ay All user interface ports are equipped with a safety interlock system which warrants laser safety These interlock devices must not be manipulated Other interfaces which are not described here are service interfaces and are only to be operated by authorized Carl Zeiss service personnel The following devices can be mounted and dismounted by the user Halogen and HBO lamp Scan head 1 14 1 Mounting and Dismounting Lamps TPMT and Switching Mirror The ports of the lamps the switching mirror and the transmission PMT are equipped with hardware interlock devices At most ports all ports of the Axioskop 2 FS MOT and the Axio Imager Z1 as well as the HBO port of the Axiovert 200 M the following interlock devices are present and have to be operated in the following way Interlock with sensor ring and contact ring Fig 1 7 Sensor ring mounted to the interface Fig 1 8 Contact ring mounted to the lamp ports on the microscope side TPMT or switching mirror The interlock is working when the sensors of the sensor ring Fig 1 7 1 are depressed by the pins on the contact ring Fig 1 8 1 Whenever this is not the case for example if the distance between the two devices is too large the laser will be blocked and the system cannot be used IEY In case the system is not operating following the removal or attachment of any device on a port with safety interlock check again the
89. a Zeiss Axiocam HRm HRc MRm camera as an Sade trac es ab al alternative external detector Beam Path and Channel Assic spectra e Click on the Config button in the Acquire eee a subordinate toolbar of the main menu The Configuration Control window opens TV ZK e Activate one of the Single Track or Multi Tube Lens o Lens 1x ay Track buttons and click on the Camera button Reflected Light The Beam Path and Channel Assignment Q panel for camera detection is opened Reflector F Hone Objective EC Plan Meofluar 40871 30 Control buttons Specimen TV Menu for selecting a display color for the camera image Transmitted Light Q Reflector Selects a beam splitter for the Or o0 excitation emission Fig 4 47 Configuration Control window Add Track Adds a second track to the camera detecting activated acquisition in Multi Track mode eg a different fluorescence filter cube or transmitted light rs If TV and LSM tracks are mixed the active detection port of the microscope has to be set according to the first track 4 66 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 3 9 LSM 5 LIVE DuoScan Panel Configuration Control The use of this function permits the use of a Channel Mode I LSM 5 LIVE or a LSM DuoScan in combination with 7 RSET a T PMT as an alternative transmitted light Senek ee l detector Listoftraks
90. a different appearance depending on which selection button has been activated Single Track and Multi Track Use the Single Track and Multi Track buttons to toggle between the two image acquisition modes single tracking and multitracking Performed settings can be stored as Track Configurations for single tracking In case the number of available channels is not sufficient for the scanning procedure further tracks can be added and contigured The combination of these tracks can also be stored as Recording Configurations for multitracking option A recording configuration may contain the maximum of 4 tracks Regardless of the number of included tracks the maximum of 8 channels incl transmission can be used in a recording configuration in multitracking If several tracks have been activated they are processed one after the other during the scan procedure If the maximum number of channels to be used in a Single Track or a Multi Track has already been achieved it is no longer possible to add further channels or tracks If a second track or further tracks are used the scan parameters can be changed as required This avoids cross talk from one channel to another when different tracks are used 03 06 B 45 0019 e 4 51 OPERATION Acquire Menu Carl Zeiss Configuration Control Channel Mode Muti Track Ratio E Spectra Single Track Beam Path and Channel Assignment LSM 5 LIVE LF 505 ca CALI BP 415 480 wre A
91. ae Set rr ene 4 325 4 15 1 Exton les Bld dela ai enep a N 4 325 4 15 2 Shut Down the WINDOWS Operating Syste ccceccccceeecccee see eeeeeseeeeeeeeseeeeeeeaneeeees 4 326 4 15 3 TANINFO OIT settresti cece cotati A N EO 4 326 4 15 4 TUNNIN Ue nO MOO kt cestcetasti oceans ccecaeasene cadsaadaeetanseanatdeenteoasetnah ened eseedcecasseneencescees 4 326 4 16 Software and Hardware Options ccccccsscseeceeseeeeeeceeseessensseessceeneessoeeseeeseeeneeessoes 4 327 03 06 B 45 0019 e 4 7 OPERATION LSM 5 LIVE Carl Zeiss Purpose LSM 5 LIVE DuoScan 4 16 1 OY IC i axa ences A eee cenconansouaianeadsorend Geugie at T O N E TETT 4 327 4 16 2 HardWare a ccascz cea cteaandanaacte pinnteseneaenstes ie AREO EEA REEE Ea i E EEr EEEE EA AiR E 4 329 4 17 Courses on How to Operate the System in an Optimized Way ccessseee 4 330 4 8 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Purpose Carl Zeiss 4 OPERATION 4 1 Purpose This chapter describes the operation of the LSM 5 LIVE Laser Scanning Microscope exemplified by typical applications in conjunction with the LSM 5 software and Its graphic user environment When starting up and operating the microscope system mind the operating instruction manuals for the Axio Imager Z1 Axiovert 200 M and Axioskop 2 FS MOT microscopes B 46 0046 Axio Imager Operating Manual B 40 080 Axiovert 200 M Operating Manual B 40 076 Axioskop 2 FS MOT Operating Manua
92. and PC table dim in mm The System rack contains all electronics and laser unit B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE DuoScan Power Requirements Carl Zeiss 2 7 1 LSM DuoScan with Ar UV Laser IEN We recommend placing the cooling unit of the Ar laser UV in a separate room to prevent heat accumulation and vibration Length of the water hose 400 cm To Cooling Unit UV LSM Power Plugs lt UV Laser Module LO Fig 2 3 Space requirements for LSM DuoScan using an AR UV Laser measurements in mm Laser Enterprise II 653 80 mW 351 nm 364 nm UV Laser module Plug in unit for external laser Fig 2 4 Lab container for UV laser setup The lab container cart 000000 0465 515 is recommended to provide space for the setup of the external UV laser the UV laser power supply and the plug in unit for external laser 03 06 B 45 0019 e 2 5 LSM 5 LIVE SETUP REQUIREMENTS Power Requirements LSM 5 LIVE Carl Zeiss LSM 5 LIVE DuoScan 2 8 Power Requirements IE The LSM 5 LIVE comes with a mains power supply cord and plug either CEE red 3 N PE 400 230V 16A or NEMA L 14 30P 2 N Ground 120 240V 30A and with the matching mains socket outlet A ground wire AWG10 green yellow is supplied because it is necessary to ground the system The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter A suitable grounding point must be installed in the room Line volt
93. and offset has to be properly tuned and MarkFirst MarkLast positions of the stack should be located approximately in the same distance from the real surface 03 06 B 45 0019 e 4 279 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan First Last e Click on the First button to calculate the topography surface by using the first slice coming from the top where the intensity reaches the value defined by the lower intensity threshold rs This mode provides better result for surfaces of semitransparent materials with inclusions of higher reflectivity or transparent multilayers with subsurface layers of higher signal intensity Extended First Last Mode 1 Definition of an intensity I threshold Starting from top bottom of a stack to find 400 _ Search of a local maximum one FWHM of actual Z PSF forwards backwards KR W N Search of the next local maximum one FWHM forwards backwards from the last max until you have not found any new local maximum 5 Last local maximum is taken as surface point Fill Holes procedure e Intensity of a missing pixel of a hole has to be interpolated by the distance weighted intensity of all Surrounding pixels e Fill hole algorithm is optimized for short calculation times 4 13 24 3 Topography Thresholds Intensity threshold Click on the Intensity button to calculate the topography surface by using the lower and the upper intensity thresholds f
94. arrow in the Button list box and window select a button out of the list rs With increasing numbers the buttons are arranged from the upper to the lower row from left hand side to right hand side e Click on the arrow in the Settings list box and select a microscope configuration e Click on the Apply button A new button with the name of the selected microscope contiguration has been created e Click on the Close button to close the Assign Microscope Settings To Button window e Click on the Close button to close the microscope window Conventional setting of the microscope Axio Imager e Click on the VIS button in the Acquire subordinate toolbar e Place specimen on microscope stage The cover slip must be facing up e Click on the Micro button to open the Microscope Control window e Via the Objective button select the required objective as follows Open the graphical pop up menu by clicking on the Objective button Click on the objective you want to select The selected objective will automatically move into the beam path e Use the focusing drive 4 29 5 to focus the required object plane e Select specimen detail by moving the stage in X and Y using the XY stage fine motion control 4 29 6 and 7 03 06 B 45 0019 e 4 45 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Switch HBO 100 power supply RL button reflected light shutter ON OFF with indicator TL button transmitted light shu
95. be entered The higher the divisor value the lower the image sharpness 4 130 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 Median filter With the median filter the gray value of each center pixel is replaced with the median value of the surrounding neighbor pixels The viewed neighbor pixels are defined by a square The moditied pixel now is the center pixel of the filter matrix The median value is defined as the middle value not average of all the gray values sorted in ascending order within a matrix Image noise will be reduced by the application of the median filter The cutoff of regions will slightly blur Local maxima will be flattened The dynamic range will be reduced considerably The settings of this filter can not be modified 5 User defined filter The User defined function permits you to create your own filters In addition to the Kernel Size the parameters Factor Divisor and Offset can be moditied here The filter result can be subtracted from the original image via the Subtract from Source check box Proceed as follows to store User defined filters e Click on the Add To List button and enter a name in the Add Filter To List window The name will be included in the Filter List Proceed as follows to activate stored User defined filters e Click on the name of the filter in the Filter List The filter will then be activated immediately Proceed as follows to delete User defined fi
96. be loaded Only the letters before the first number are stated Input Name of the resulting image in which the image sequence will be saved Save Image As This function saves an image or image sequence to disk or network drive Gave Image As Ea File CALSM51 04 3D forLemImages S ensor elh lem File Path lsm CALSM51 0430 forLems imagesS ensc EJE EJLSMS10 EJ 3DforLsm EJ Images oe SenzorCell Files of Type Drive LSM5 Images lsm E c Fig 6 7 All the files in the current directory that have the selected image format are listed in the File Name list box The directories of the current drive are listed in the Directories list box Use the Drives list box to choose a different drive Use the list box Files of Type to select the image format Currently the LSM image format lsm and the Carl Zeiss Vision file format KE Images 0 img is supported 6 12 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss By choosing the Carl Zeiss Vision file format KE Images 0 img two files per channel are saved On one hand the Carl Zeiss Vision type image sequence file on the other hand the file with the image properties One pair of files is written per channel They are numbered automatically starting with zero A one number digit is added to the end of the filenames The two files share the same filename but have different filename extensions img and 3d The content
97. button for data flow into the program flow Action blocks can be removed using the Delete key of the keyboard 3 Without using the Image Display action block in the data flow column the result of the scan will not be displayed see also description of Scan and Time Series action blocks The work flow is generated by connecting the action blocks using the mouse cursor e Click onto an output arrow with the left mouse button hold down the mouse button and drag the mouse cursor away A connecting line appears e Drag the line to the input arrow of another action block Only arrows of the same color can be connected According to the action blocks a set of properties is listed which can be assigned with free or predetined values A set of defined properties is assigned to one block only If this block is used more than once the properties can be set differently Clicking the Read Back button will take over the current settings from the LSM main program for the displayed properties list e Mark the check boxes next to the parameters individually or click Use all or Use none to check or uncheck all parameters with one click e For changing a values click into the Value column 4 190 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss It is possible to define variables A variable has a property name and a mathematical value The Assignment action block can be used to assign a new value to a vari
98. button Activates the circle drawing mode Clicking and holding down the mouse button sets the center point drag the diameter and release the mouse button to end the procedure So We Recycle bin button All the ROIs to the image are deleted Rectangle button Activates the rectangle drawing mode Click and hold down the mouse button drag the rectangle in any direction release the mouse button to end the procedure Ellipse button Activates the ellipse drawing mode The first click sets the center point the displayed line permits the determination of the first dimension the second click sets the first dimension the second dimension and the rotation direction can then be determined the third click sets the second dimension and the direction and ends the procedure iS if le 03 06 B 45 0019 e 4 313 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Polyline button Activates polyline drawing mode The first click sets the starting point each additional click adds a further line a double click on the starting point closes the figure and ends the procedure Line button This button allows you to determine the line thickness of the ROI outline Color Auto button One color from the list of colors can be assigned to all ROIs When Auto is pressed the outlines of all ROIs are automatically colored differently es _ ES Q 02 Buttons for diagram display 1 Chan AO Mono Area Me
99. check box to close the Filter Preview Image Display window I After a change of the filter settings click in the Filter Preview Image Display window once to update it 4 6 8 Contrast The Contrast function permits the subsequent modification of contrast and brightness of the stored image e Open the image to be processed and click on the Contrast button The function is performed with firmly set parameters and the result is displayed in a new Image Display window The procedure can be repeated as often as required e The newly created image can be stored using the Save As function 4 152 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 6 9 Interpolate This function permits the continuous contrast and brightness change in a Z stack or Z stacks over time through interpolation between the starting and end values This permits the subsequent compensation of signal loss when imaging further into deeper tissue regions where the excitation efficiency decreases but also the detection efficiency Interpolation can be defined for the entire image or only for individual channels L The Modify Series macro can be used to convert time series data for use of the Interpolate function 4 6 9 1 Open Close the Interpolate Brightness and Contrast Window e Click on the Interpolate button in the Process Subordinate toolbar of the Main menu This opens the Interpolate Brightness and Contrast window e Click on the
100. configuration A no longer required recording configuration can be deleted as follows e Click on the Config button the Recording Configurations window appears on the screen e Select the configuration to be deleted from the Configurations list box e Click on the Delete button e Close the window by clicking on Close 03 06 B 45 0019 e 4 603 Carl Zeiss 4 5 3 6 Transmitted light preset 7 Configuration Control Damage Mode TUE anaemia Beam Path and Channel Assignment Fig 4 46 Transmitted light preset 4 64 OPERATION LSM 5 LIVE Acquire Menu LSM 5 LIVE DuoScan To add a transmitted light image the light level of the Halogen lamp can be preselected in the configuration control window The illumination is activated when the actual scan is started To separate the transmitted light signal trom the emitted fluorescence choose e g the BP 700 750 in Ch1 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 3 7 Ratio Settings Panel The Ratio Settings panel permits you to activate two additional Ratio channels e Click on the Ratio button The Ratio Settings panel is displayed at the bottom of the Configuration Control window The settings of the selected tracking mode Single Track Multi Track remain unchanged e The Ratio Settings panel is only available in the Single Track and Multi Track mode The following function elements are provided in the Ratio Settings panel
101. connection of the Contact ring to the Sensor ring 1 20 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan User Interface Carl Zeiss For dismounting the lamps TPMT or switching mirror slightly unscrew the contact ring first Fig 1 8 2 or Fig 1 9 2 so it can be moved away from the sensor ring Fig 1 9 1 Then unscrew the lamp TPMT or switching mirror main screw Fig 1 9 3 which is in one of the recesses of the sensor ring Fig 1 7 2 Hold the device to be dismounted with one hand while unscrewing to keep it from dropping The now empty port has to be closed with the blind cap to restore the functionality of the system Use the main screw of the port to fix the cap Make sure the pins of the cap depress the sensors of the sensor ring L Do not remove the sensor ring from the microscope This might result in failure of laser safety and a non operating system For mounting any lamp TPMT or the switching mirror back onto the microscope reverse the steps for dismounting the device Be careful not to bend the pins on the contact ring when screwing the device onto the microscope port For the Axiovert 200 M transmission port and the two ports available on the motorized switching mirror no sensor ring is present Instead the sensors are directly installed at the Axiovert 200 M transmission port or the two ports of the mo torized switching mirror 03 06 B 45 0019 e Fig 1 9 HBO lamp mounted to Axiove
102. definition of area for size and intensity measurement Copies area values to the clipboard only available if the Area button is activated B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 20 2 Area Function e Click on the Area button in the Histogram toolbar The function elements for Area measurement are displayed at the bottom right of the Histogram toolbar Oe bepa p ee es Bs sie hurai bere Sipe r sil ia gees a e F Teri Mids TEH chare 1b tia Daip a 1 a i Pi Fig 4 262 Image Display window Area Measure display The following function elements are available Step Low High Ch51 T1 7 03 06 Set the number of intensity steps which shall be displayed in the diagram Step 1 corresponds to 256 intensity steps Step 64 to 4 intensity steps in case of 8 bit images Reduction is made by averaging Threshold low slider with Color selection button The intensity values below threshold are not displayed The removed areas are masked in the color selected in the Color selection button Threshold high slider with Color selection button The intensity values above threshold are not displayed The removed areas are masked in the color selected in the Color selection button Ch1 Ch3 buttons Selection of the channel for which the area measurement is to be performed Arrow selection button Activation of the mouse button for selection
103. direct sunlight To avoid heat build ups the ventilation slots on the microscope system must not be covered up The system must not be set up in areas with potential danger by explosives The unit must be connected to a properly installed socket outlet with earthing contact by means of the mains cables supplied Continuity of PE connection must not be affected by the use of extension leads The system contains components with dangerous voltage The system must not be opened by anybody else than authorized Carl Zeiss Service staff Before opening the main plug has to be disconnected Before connecting the mains cables please check whether your mains voltage corresponds to the voltage specified on the rating plate of the laser module For reasons of laser safety all ports must either be equipped with the corresponding device scan head camera HBO lamp etc or covered with the counterpart of the laser safety kit provided gt gt gt 1 12 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Protection against white light Carl Zeiss A gt e b D Maintenance repair modification addition removal or exchange of components or other interference with the equipment beyond the operations described in this manual may only be carried out by the manufacturer Carl Zeiss or by persons expressly authorized by Carl Zeiss to do SO This applies especially to the microscope system the laser scanning module las
104. display is red busy B 45 0019 e 4 219 OPERATION LSM 5 LIVE Carl Zeiss Maintain Menu LSM 5 LIVE DuoScan Camera Color Adjustment 4 10 4 Camera eset In the Camera Color Adjustment window the user can adjust the white balance and color balance of a connected camera Clicking the Pic button allows to set the white balance using the mouse cursor in the camera image Using the arrow buttons to adjust the color balance of the camera T Enhance Fig 4 232 Camera Color Adjustment window 4 10 5 Reboot The Reboot function is for servicing purposes only and may only be performed by authorized personnel Its access Is therefore password protected 4 10 6 HW Admin The HW Admin function is for servicing purposes and may only be used by authorized service personnel Its access Is therefore password protected 4 10 7 Test Grid The TestGrid function is for servicing purposes only and may only be performed by authorized personnel Its access Is therefore password protected 4 220 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Window Menu 4 11 Window Menu The Window menu includes the additional Full Screen functions Full Screen Close All Image Windows Toolbar and Scan Information which are not available from a toolbar Close All Image Windows Scan Information Fig 4 233 Window menu 4 11 1 Full Screen This function shows the active Image Display window in full screen size e Activa
105. e Close the LSM 5 software e Switch on the power supply of the Enterprise power potentiometer turned to maximum e Start the LSM 5 software again 4 5 2 Microscope Control The Microscope Control Micro button window permits motorized functions objective and reflector change condensor filter and diaphragm settings and the illumination mode transmitted or reflected light of the connected microscope to be controlled via the software Without any difference to software control these microscope functions can also be operated directly on the stand via the relevant controls In that case any changes are recorded by the software and displayed in the relevant windows panels 4 5 2 1 Open the Microscope Control Window e Click on the Micro button This opens the Microscope Control window on the screen 4 40 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss After conclusion of the conventional setting of the connected microscope the Microscope Control window can be closed again e Click on the Close button in the Microscope Control window The Microscope Control window will be closed 4 5 2 2 Microscope Control Window for Axio Imager Z1 e Click on the Micro button in the main frame e The microscope window opens in the last saved configuration e By clicking on the More Less button the microscope window is displayed with or without detailed microscope beampath panel Reflecte
106. element is used that generates a closed contour Color selection box The colors displayed in the Color selection box can be assigned to the overlay elements with a click of the mouse The currently selected color is displayed in the larger rectangle left top of the selection box A selected color is automatically assigned to the currently selected overlay element and then to all the elements created afterwards B 45 0019 e 4 233 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 7 Select Contr This function allows to change the contrast and brightness of an image change the contrast and brightness of a channel of an image define interactively a new relationship between the intensities of pixels in the image memory and the displayed values of this pixel intensities on the computer screen Click on Contr will display the Contrast toolbar Any changes done with this toolbar are effective immediately Moditication done by this function are for display purposes only To permanently change the contrast and brightness of an image use the function Contrast in the Process menu Fig 4 243 Image Display window Select Contr e Click on the Contr button in the Select toolbar The Brightness and Contrast window will be displayed e Change brightness and contrast via the sliders in the Brightness and Contrast window You can adjust each channel individually by activating
107. ending continuous averaging is displayed instead of the Cont button Use the Single button in this case to start continuous scanning When you click on the Finish button the scan currently in progress will be completed before the process is stopped Zoom Rotation amp Offset Zoom Rotation amp Offset panel oom In this panel the scan range is set for zoom and offset in relation to the field of view of the microscope The diagonals of the outer square on Offset the right hand side correspond to the field of view of the microscope f The inner square contained in it rectangle in the ae a im case of differently set frame size represents the scan range and immediately shows the changes Fig 4 55 Zoom Rotation amp Offset panel made to zoom and offset The blue line at the top of the scan range is helptul for orientation when the scan range is rotated in the direction of the field of view e Set the desired zoom factor via the slider Zoom or by clicking on the arrow buttons The zoom factor can be set continuously in the range from 0 5 to the maximum of 2 0 and is displayed in the relevant input box The value 0 5 corresponds to factor 1 and value 2 0 to factor 4 related to the field of view of 18 mm in the intermediate plane Clicking on button 1 enables immediate resetting to the zoom factor 1 Recommended setting to start with Zoom 1 e Move the scan area by clicking on the 4 arrow buttons Of
108. etc are supported LB When stacks or time series are exported each frame is stored as an individual image Export Images and Data El ES Channels H Chst T G chs2 T E Eh2 T1 Palette image Image tepe ay data single plane Speichern E3 Spectral mdb wl fo m3 mE Dateiname Dateityp Tagged Image File tif Abbrechen ME Fig 4 22 Export Images and Data window 03 06 B 45 0019 e 4 33 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan T en 4 4 7 Multi Print a Z This function permits you to arrange several images on one print page and to print them out together e Click on the Multi Print button in the File subordinate toolbar of the Main menu i T Oy P i pil w oO D n Qo pi oO This opens the Print AIM window Q a i The main area of the Print AIM window is used for the display of the print page in the selected paper orientation and for the arrangement of the l images to be printed The Print toolbar with the following buttons is displayed on the right hand side of the window Paste Paste from clipboard to sheet Delete Delete marked image Print Start printing Setup Printer setup FIOLE PUNURAIM indo Landsc Landscape paper orientation Portrait Portrait paper orientation Zoom slider Zoom function for page preview 4 4 7 1 Overlay Toolbar The following funct
109. even illumination in conventional microscopy the LSM technique projects the light of a point light source a laser through a high NA objective onto a certain object plane of interest as a nearly diffraction limited focus However if not for another trick the stray light produced outside the object plane or the fluorescence of fluorescent specimens would disturb the in focus image of object point of interest resulting in a blurred image of poor contrast The problem is therefore how to capture only the light coming immediately from the object point in focus while obstructing the light coming from out of focus areas of the specimen e The light reflected or the fluorescence light produced at the focus of the high NA objective is projected onto a variable aperture diaphragm by the same objective and a tube lens The focus inside the specimen and the aperture are situated at optically conjugate points confocal imaging The decisive advantage of this arrangement is the fact that essentially no other light than that coming from the object plane of interest can pass the narrow aperture and be registered by a detector Unwanted light coming trom other specimen areas is focused outside the aperture which passes only a small fraction of it The smaller the aperture the less stray light or fluorescence from out of focus areas will get on the detector The image point thus generated is largely free from blur caused by unwanted light e In o
110. follows to select an image via the image selection box e Click on the arrow button The image selection box is opened and all the currently loaded images are displayed in a minimized form e Click on the required image This image will then appear in the display box of the image selection box and has been selected as Source 1 Use the Click into window button to directly select the opened image e Click on the Click into window button first and then double click on the relevant Image Display window The selected image will then be displayed in the display box of the image selection box and has been activated as Source 1 The channel which is to be used for the Add operation is selected via the Channel selection box e Click on the arrow button The Channel selection box is opened and shows all the recorded channels of the relevant image e Click on the required channel to activate it In the Source 2 panel the second image source for the addition process is determined The procedure is identical to that for Source 1 e Select the image for Source 2 and the relevant channel Destination 4 6 1 3 Destination Panel In the Destination panel it is determined in which Image Display window the Add operation is Channel New performed and the data format which the newly created image shall have New Image 3 bit Fig 4 109 Destination panel The Add operation can be performed in an already opened window or in a new Image
111. for five minutes before Fig 4 215 Hardware tab switching off the system Light manager activates or deactivates the light microscope stands light manager Shutter filter wheel activates or deactivates additional laser attenuation by grey filters LSM 510 only Simultaneous grab and bleach activates or deactivates the simultaneous grab and bleach option in the bleach menu This function can create fluorescence artifacts depending on sample properties and confocal aperture setting it is recommended to close the confocal aperture to 1 airy unit geccecccccccccccccesescccccccecccecececcecececsesesesssssssesssse 4 9 2 16 Ima ge Dis p ay Program Start Hardware _ _tmage Display Save m Display Window Toolbars Palettes color The Image Display tab enables you to determine M Show Channels Toolbar V Switch to mono on activation of a palette f i Show Zoom Toolbar Switch to color on deactivation of a palette the window toolbars which shall be automatically T Show Slice Toolbar eee displayed when an Image Display window is ala Rep E E N O p ene d Diagram background color C Furthermore the color mode color mono to which the image display will switch when the Color Palette function is opened closed can be determined Fig 4 216 Image Display tab 1 Display Windows Toolbars On activation of the relevant check box the following window toolbars are automatically displayed when an Image Display window is opened
112. generated by the Zeiss LSM software This can be three dimensional image data or a time sequence of two dimensional images slices Each slice as well as the sequence can consist of up to eight Single slice with channels An image sequence consists of a series multiple channels of individual 2D images and has a name that designates the entire sequence In general an image sequence is handled as a single object in the Single slice with single channel Image sequence system Individual channels or slices can be Multichannel addressed Positive rotation directions of the axes The following terms and definitions apply for the 3D for LSM software An image sequence is a number of individual sequential images usually called slices in the dialog boxes the spacing between which is equal Image sequences can contain up to 12 bit of image data per channel A sequence slice can consist of up to eight channels Fig 6 1 The maximum size of an image sequence is limited by the provided memory of the operating system A voxel is the smallest element of an image sequence the equivalent of a pixel in a 2D image All voxels in a given image sequence are the same size The coordinate system originates in the left upper front corner of the image sequence This point has the coordinates O O 0 All angles are positive for rotations to the right in the direction of the positive coordinate
113. gt Interval prn 1 CS Ss gt Current Slice 11 A gt C Keep Interval Keep slice Fig 4 64 Scan Control window Z Sectioning tab activated B 45 0019 e 4 81 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan e Click on the Range button The XZ scan will be performed and displayed in the Image Display window At the same time the position of the current slice is shown with a green line and the positions of the first and last slice with two red lines Red lower lt range limit Fig 4 65 Scan Control window and Image Display window e Moving the green line current slice enables you to change the current focus position moving the Stage or nosepiece in the process The stack limits are also changed while interval and Num Slice remain unchanged e Shifting one of the red lines enables you to change the stack size in that case the interval size is matched and the Num Slice remains constant Changing the values of Num Slice Interval and Current Slice in the Z Sectioning tab will of course also change the positions of the red and green lines in the Image Display window e Aclick on the Start button will start the recording of the Z Stack The settings of the entire Scan Control window Mode Channels Z settings will be used when the stack Is produced fs In Frame mode the stack is acquired as a stack of images in xyz In Line mode the stac
114. humidity lt 65 at 30 C 8 Operation altitude max 2000 m 03 06 B 45 0019 e 1 17 Carl Zeiss 1 12 NOTES ON DEVICE SAFETY LSM 5 LIVE Notes on Handling the Computer and Data Media LSM 5 LIVE DuoScan Notes on Handling the Computer and Data Media The computer used as standard in your LSM system is an IBM compatible high end Pentium computer with WINDOWS XP operating system R Do make sure though that you receive your LSM system with the operating system installed with initialization and start files set up and with the LSM program also installed When working with the hard disk it is important to know that the more data it contains the slower its operation will become Therefore data that you do not need permanently should be stored on other external devices When handling diskettes avoid data losses by protecting them against extreme temperatures moisture and magnetic fields The data on a diskette is stored in the form of magnetic signals To some extent monitors telephones or even lamps generate magnetic fields that might destroy this data Also never open the metal cover on diskette cases A diskette s surface can also be destroyed by touching it When handling CDs CD ROMs or DVDs do not touch the data side of the disc the side of the disc with no label or printing Do not apply paper labels or write on any part of the disc data side or label side If dust or fingerprints get on the disc wipe it with a s
115. mdb AIM Iols Record 5 ofS Mame Date f Time scan Mode Pixel Depth stack Size stack Pixel j pmj O 042005 02 52 48 PM Plane original data 512x512 x 920 9 um x92 01 04 2005 02 52 48 Phi Plane original data 512x512 x1 920 9 um x 92 01 04 2005 03 29 36 Phi Plane original data 256x 256 x 1 920 9 urn x 92 4 Total 1 471 MByte Selected 145 497 kByte Current 145 497 kBute Clipboard 337 063 kByte Fig 4 13 Database window Table display mode In the Options menu in the function Settings it is possible to define the start mode of the image database Form Gallery Table the recordset displayed first last middle and the parameters shown e To select one of the images of the database for normal size presentation double click on the desired line The same can be achieved by clicking on the desired image in the table and then clicking on the Load button If several images have been selected they will all be opened and displayed one after the other 3 _ The selection of several images is performed in the same way as in the Gallery mode i e by pressing the Shift and Ctrl keys 4 4 3 4 Load an Image into the Image Display Window e Click on the Load button to load the selected images into the Image Display window or double click on the appropriate image in the database window 4 24 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss 4 4 3 5 Load Image with
116. must be selected This function is especially well suited for masking images If one or both input sequences are multichannel sequences any number or combination can be selected The number of selected channels for Input 1 and Input 2 must be the same They will be combined from left to right Parameters Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence Boolean Or This function carries out a bit by bit Or calculation for the images Input 1 and Input 2 Boolean And r or Not Mask Input 1 hooo A Input 2 pooo A Output Booo New OE Cancel Fig 6 27 The Or tab sheet of the Boolean dialog window must be selected This function can be used to combine binary masks or regions 03 06 B 45 0019 e 6 37 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan If one or both input sequences are multichannel sequences any number or combination can be selected The number of selected channels for Input 1 and Input 2 must be the same They will be combined trom left to right Parameters Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence Boolean Xor This function carries out a bit by bit Xor calculation for the images Input 1 and Input 2 Boolean And Or Xor Not Mask Input 1 fo Alf 1 Input 2 pooo A Output Booo New UE Cancel Fig 6 28 The Xor option button of the
117. name The Apply button permits existing stored microscope contigurations to be loaded The Delete button permits existing microscope configurations to be deleted The Assign button permits the assignment of a microscope contiguration to a button Note Depending on the microscope configuration settings must be done manually if necessary 03 06 74 Microscope Control Microscope Settings Close GFP Apply Store Delete Assign Button GFP Ly Rhod Trans on Less G Fig 10 Mun Condensor Transmitted Light w Aperture 0 0529 Off O 0 Objective Plan 4pochromat 631 4 Oil Reflected Light Reflector GFP Ss Tube Lens Lens LSM Microscope Control window e g Axiovert 200 M Microscope Settings Dar Apply Store Delete Assign Button DAPI Close FITC TRITC E a Ae Transmitted Light Q Transmitted Light ne E Pee On a S Fig 11 Mone Tube Lens Lens LSM Microscope Control window with Transmitted Light pop up menu Configuring the beam path and lasers 4 Configuration Control e Click on the LSM button in the Acquire subordinate toolbar for laser scanning Channel Mode datio hline Fingerprinting Single Track Multi Track Ratio E Choosing the configuration Beam Path and Channel Assignment _ gt pectra LSM DuoScan LSM 5 LIVE Use for single double and triple labeling LP S05 m simultaneous scanning only yo A
118. of the shown channels can be set with the function Set Channel Colour Parameters None Exit This function terminates the application completely All images and image sequences shown in the Gallery will be deleted trom the memory Save those images which might be used for any further processing Parameters None 6 3 2 Functions in the Edit Menu Copy This function copies the current Display window contents to the clipboard No dialog is shown Before the execution of this function any image or image sequence can be selected to be displayed From a multichannel sequence any channel status on or off combination can be defined The colours of the shown channels can be set with the function Set Channel Colour The current zoom factor of the Display window is not taken into account the image is copied without any zoom The image is copied as a true colour image with 24 bit resolution Afterwards the contents can be pasted to any other Windows application Parameters None 6 14 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Edit Channels This function allows to add or to remove channels to a single or multichannel image On the Add Channel tab sheet the channels of different Input sequences can be defined to add combine channels to an Output sequence Edit Channels Add Channel Delete Channel Input 7 i fafa 2 fa Input 2 Boo A Output 2 New Outpu
119. of the recovery can be calculated using the following formula Am a itn Ig fl i I 7 i fi i 15 nj 4 Tera ial Chi i DaT i a m bile Fochen Te mma bele mactan 1514 325802 1345 EIE 1 674962 ER AREH Read Skils 13 1 chene 12 bf Heppa Pig Pag hom image File Fig 4 141 Image window displaying the analysis of FRAP data using a mono exponential fit If the analysis is done using the double exponential fit the fitted curve displays the mean of the fitted values for the two different mobile fractions The table shows the following additional parameters 03 06 B 45 0019 e 4 149 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan is The amplitude of the two curves displayed as one which equals each part of the mobile fractions 11 and 12 The fitted parameters T1 and T2 seconds for each mobile fraction The rate constant for the exchange of molecules between the bleached region and the surrounding area K1 and K2 per second for each mobile fraction PML Bodies 007 AIM lol x Kinetic Analysis Diagram Intensity Display 0 8 ee Oe gan ae F 06 Chan 3 ROI 0443 Style 0 2 IV Normalize Table 0 5 10 15 20 25 Time s Copy Results Ch2 T1 x Ch2 11 fit i0 i1 Ti KI I2 T K2 i delta Save Resuks mobile fraction s l s mobile fraction s l s immobile fraction SSI 1 000000 0 598690 1 2198
120. on the screen e On the Channel Settings panel of the Scan Control window set the CCD detector gain with the Detector Gain slider The image should not have more than a trace of red This adjustment is very sensitive Try using the left and right arrows to make the adjustment instead of dragging the slider bar Feri TOS x TOG Z chirii bet Fa pepe dota Depi Zoe 1 _ Fiabe Abo Palia Fig 4 313 Image Display window 4 320 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan e To adjust the black level background use the Ampl Offset slider so that areas without picture content just show a trace of blue e In the Color Palette List panel of the Color Palette window click on No Palette This deselects the Range Indicator and activates the new presentation e In the Scan Control window click on the Stop button This stops the continuous scan i Ifyou use the Range Indicator for image optimization it may happen that the ranges marked in the Range Indicator will vary when the channel color is changed 4 14 1 3 Exposure Time Scan Average and Pixel Depth The signal to noise ratio can be substantially improved by reducing the scanning speed to an acceptable level and averaging over several scans i e with an average Number greater than 1 for the Mean average Method in the Scan Control window e Use the Exposure Time slider in the Objective Lens Image Size and Line amp Step Factor p
121. opens the Visual Macro Editor window e Click on the I button in the upper right side of the window to quitfamy362 Fig 4 190 Visual Macro Editor window 03 06 B 45 0019 e 4 189 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan 4 8 9 2 Function Description The Editor uses predefined action blocks The action blocks for setting up the work flow of the Macro are categorized according to their properties The following categories are displayed on the left hand side of the Visual Macro Editor control window Acquisition Windows Load Save Program Flow Process Hardware Control e Click on one of the Category selection buttons will display the available action blocks The action blocks can be positioned according to their function either in the Program Flow column or the Data Flow column The color of the connection arrows of the action block defines the possibility to position the block in the program flow column or the data flow column s e Click at the action block with the left mouse button hold down the mouse button and drag the mouse cursor into the flow column Release the mouse button Action blocks for the program flow show red connection arrows VW as well as blue output arrows Vv to connect to the data flow Action blocks for the data flow show blue connection arrows W only i It is not possible to drag an action block for program flow into the data flow or an action
122. operation of the LSM 5 LIVE IEN Read these operating instructions and all device publications belonging to the system conscien tiously before operating the LSM 5 LIVE You can obtain additional information on the hard ware configuration delivered and on optional system extensions from the manufacturer or via the service hotline The LSM 5 LIVE has been designed built and tested in conformity with the following regulations and guidelines DIN EN 61010 1 IEC 601010 1 Safety requirements for electrical equipment for measurement control and laboratory use DIN EN 60825 1 IEC publication 60825 1 Safety of laser equipment taking relevant CSA and UL specifications into account DIN EN 61326 Electrical equipment for control technology and laboratory use EMC require ments Low voltage directive 73 23 EWG EMC directive 89 336 EWG The company works according to a certified Environment Management System according to ISO 14001 The Product was developed tested and produced in accordance with the valid regulations and guidelines for environmental law of the European Union The product and its accessories have been classified as instrument category 9 laboratory equipment or comparable standard The product and its accessories agree with the EU regulations 2002 95 EG RoHS and 2002 96 EG WEEE if applicable for the product Carl Zeiss has installed a process for taking back and recycling the instruments within the me
123. out Selection of the trigger key 1 4 for the out signal list box 1 7 Setting marker for the current scan e A marker for the current scan Is set by clicking on one of the Set 1 to 7 marker buttons The assignment of any required comment for the marker must be performed as follows e Click in the Edit Text box of the required marker key e g Set 1 to open the editing box e Enter the comments via the keyboard Then click outside the editing box to close this box again You can assign comments of any required length to all the seven Set buttons and store this assignment as a combination of marker keys e Enter a name in the Marker list box and click on Store to store the combination If required a combination of markers can be activated again quickly e Open the Marker list box with a click on the arrow button and select the required combination with a click of the mouse e Then click on the Apply button to activate the combination The relevant comments are displayed in the Edit Text boxes of the Set buttons Combinations which are no longer required can be deleted e Open the Marker list box and select the required combination e Click on the Delete button The combination will be removed The marker buttons can also be activated via keys 1 to 4 of the Trigger Control e Click on the required Set button e Open the Trigger in list box with a click on the arrow button e Select one of the trigger keys 1 to 4 e g Trigger4
124. page T m 4 274 Specular E fioo Shininess 25 Projection View ange 45 0 Distance 1 po Fig 4 292 3D Rendering window Light panel Determines the properties of illumination on a sample Distance Goes for diffuse and specular see visualization Azimuth See visualization Rise angle of the sun Elongation See visualization North south east west direction of the sun Background Choose background color Material panel Determines the reflective properties of a sample Ambient Specular Material properties how many of the light component are projected by the material into which spectral ranges Shininess Goes together with specular light Shininess equal to 25 determines diffuse light 03 06 B 45 0019 e 4 291 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Projection panel Determines the reflective properties of a sample View angle Determines the perspective 0 0 parallel projection central projection Distance Zoom function Zoom in zoom out tip trace AlM Eie x Display Topography Generate gt 2 First Maximum Center Threshold p gt Intensity Height Auto Z None f Median FFT a Geometry 4t 5 Inverse Fit Manu Tilt Display P 2D lIso Lines Measure Off Diagram Roughn Volume Profile Dist _ T2 Fill Level 7 gt Ee Ready 1024 x 1024 x 36 1 channel 12 bit
125. pour slide object Continue with nest Next Cancel Objective Control window TW Pinhole and Collimator Close Pinhole LIVE_PH2 Description LIVE Pinhole Ch2 Diameter pm 7 1 q m gt Pixel Shitt s dm gt Position r Add al SS a gt Store Curent a Move To Stored Tl Laur oe Adjust Sey Mell Automatically T Fast Adjust Mode Collimator Collimator LIVE Ay Description Collimator LIVE Ay Position mm 455 Ki Hie gt Store Current pia Move To Stored Position Position Move io pte Roster Adjustment Mo objective data available Pinhole and Collimator window 03 06 B 45 0019 e 4 215 OPERATION LSM 5 LIVE Carl Zeiss Maintain Menu LSM 5 LIVE DuoScan 4 10 3 1 Open Close the Pinhole and Collimator Window e Click on the Pinhole button in the Maintain subordinate toolbar of the Main menu This opens the Pinhole and Collimator window e Click on the Close button to quit the window 4 10 3 2 Pinhole Panel No further software function can be activated and executed during aperture adjustment Pinhole Description Selection of apertures PH1 to PH2 to be adjusted via the Pinhole selection field box display of the relevant channel in the Description field Diameter Pixel Shift Setting of size pixel shift and Y position of the aperture in relation to the Position Y slider beam path using the slider or arrow buttons status display for setting proce
126. procedure shall start End Image E z Last Clicking on the First button permits the fast Brightness fe selection j the first slice in the u Contrast A m ai _ ay cena and Contrast sliders to set ge brightness and contrast for the first Ch1 ae ee All slice Start Image F Overwrite Source Images e Use the End Image slider to select the slice at T Ignore Images less than Start Image and greater than End Image which the interpolation procedure shall end Clicking on the Last button permits the fast ROgeee iMtErpOANON pane selection of the last slice in a series e Use the Brightness and Contrast sliders to set the image brightness and contrast for the last slice End Image e Use the available Channel buttons e g Ch1 to select the channel for interpolation or click on the All button if the entire image is to be interpolated e Having set the parameters click on the Apply button Interpolation will be performed in a new Image Display window e The newly created image series can be stored using the Save As function LS If you activate the Overwrite Source Images check box interpolation will be performed in the current Image Display window If you activate the Ignore Images less than Start Image and greater than End Image check box only the slices lying between Start Image and End Image will be taken into consideration for interpolation Otherwise brightness and contrast will also be changed f
127. reflector module filter sets to suit the type of fluorescence excitation Proceed as follows e Click on the reflector turret button e Click on the desired reflector module The reflector turret moves the selected reflector module into the beam path i The FITC filter set consists of an excitation filter for the 450 490 nm spectral range an FT color splitter for 510 nm and an LP long pass filter which passes emission light wavelengths greater than 510 nm FSET 09 FITC FSET 15 Rhodamine FSET 01 DAPI Other filter sets DAPI BP 365 FSETO1 FT 395 LP 397 Rhodamin BP 546 FSET15 FT 580 LP 590 i The filter sets described in this section are included in the standard configuration but other sets are available on request iS Ifthe X Cite 120 fiber coupled lamp for reflected light illumination is used a aem further dialog is opened when clicking the EEN Close Reflected light button The lamp internal iemel ounn 1 shutter and the attenuation function can Ei i 2 be controlled via the LSM Software The lamp has to be switched on first using the main switch on the lamp housing Remote On Fig 4 30 X Cite 120 control panel 03 06 B 45 0019 e 4 45 Carl Zeiss Remote ON Shutter Intensity Lamp Hours OPERATION LSM 5 LIVE Acquire Menu LSM 5 LIVE DuoScan Enables the attenuation control via LSM Software This is also enabled when clicking the ON button Opens the shutter in the reflected
128. software controlled using the Stage XY buttons or manually The Current Position display for X and Y is below Below that you will find the Marks selection box of marked positions and the possibility to activate and delete them Moving the scanning stage Stage XY buttons e Clicking on the arrow buttons moves the stage in X or Y direction e Clicking on the Center button moves the stage in the XY 0 position XY Step slider 1 um is the smallest value which can be set for XY movement and 100 um the highest Manual check box This check box activates deactivates the motor control of the stage and the joystick if available If Manual is active the scanning stage can be moved manually via the knurled screws The Move To and Center function buttons in Stage Position are without a function The Current Position is updated You can zero the display via ZERO and mark manually set positions Mark pos The scanning stage cannot be moved via the software or the joystick If Manual is deactivated the scanning stage can be moved via the software or the joystick All the functions of the Stage Position window are available Current Pos ition field Current Pos displays the currently set stage position in relation to the zero position Marks selection box Clicking on the arrow button displays the table of the session related marked specimen areas The table includes the ordinal number the X position and the Y position
129. the list Selected Features can also be transferred by clicking on the button in the middle lt lt gt gt of the dialog The combo box above the right list represents predefined feature lists Selecting one of the entries will Till the right list with these features previously selected features will be overwritten The button Select All will copy all features to the list of selected features The button Remove All will clear the list of selected features Clicking on the Apply button will execute the measurement process and switch to the General tab sheet of the dialog Parameters Available Features List of available object features Selected Features List of selected object features Select All Select all available object features for measurement Remove All Remove all object features from the selected features list Volume Features geometric The volume related measurement generates one measured value per image sequence The following table contains the predefined volume characteristics Group Name Name Description Count VolCount Number of regions measured Volume VolVolume Total volume of all regions Volume Percentage VolVolumeP Total volume of all regions in relation to the volume of the image sequence Volume Features densitometric Group Name Name Description Surface Area VolSurfArea Total surface area of all regions Mean Densitometric VolMeanD Mean grey value of all regions Standard Deviation VolStd
130. the relevant specifications Introduction to Laser Scanning Microscopy Here you will find an introduction to Laser Scanning Microscopy with an explanation of the principles of confocal imaging The section also outlines the ways to present LSM image series in three dimensions and introduces you to the performance features of your LSM 5 LIVE Operation In the Operation section you will find the most important steps and procedures of the LSM menu structure The step by step description how to get an image will be shown by typical application examples including the WINDOWS XP graphic user environment Tools This section contains a description of the use of the tools for setting the microscope 3D for LSM This section contains a description of the LSM 3D software package basic program and add ons At the same time all functions and settings are presented in a systematic form and in the order in which they can be reached trom the basic menu via sub menus and dialog boxes Annex The annex contains the Application specific Configurations special notes and information for using the LSM microscope and the brochure The confocal Laser Scanning Microscopy Certification Brief Operating Manual This section contains the brief instructions of the LSM 5 LIVE Microscope Systems Laser safety warning labels B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Contents Carl Zeiss CHAPTER 1 NOTES ON DEVICE S
131. to use a different laser other than the ones above If used properly the LSM 5 LIVE will not pose any laser radiation risks for operating staff Nevertheless you should observe the following warnings 03 06 B 45 0019 e 1 15 Carl Zeiss A NOTES ON DEVICE SAFETY LSM 5 LIVE Notes on Handling the Laser Components LSM 5 LIVE DuoScan e f necessary insofar as specified by law inform the laser protection officer before commis sioning the laser e The laser modules are equipped with a key interlock e Always store keys for laser key switches and if applicable keys for further laser power supply units where they are inaccessible to persons not authorized to operate the laser e A red LED on the front of the scan head lights up when one or all of the lasers are switched on e Do not place any reflecting objects into the beam path e Never open any covers or panels e Never look into the laser beam not even to simply view the specimen whether with the aid of optical instruments or without Otherwise you risk going blind e Do not leave any empty objective positions of the nosepiece uncovered Suitable protective measures must be taken if gases dust or vapors hazardous to health secondary radiation or explosive objects should arise on the specimen as a result of laser radiation B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Notes on Handling HBO and X Cite Light Sources Carl Zeiss 1 9 Notes o
132. tracks with regard to channels emission filters and dichroic beam splitters e Click on the Multi Track button The Configuration Control window for multitracking appears which means that the List of Tracks panel is additionally displayed The tracks required for multitracking can either be configured manually one after the other identical to single tracking and then stored as recording configuration or already existing recording configurations can be used and changed as required It is also possible to load already stored track configurations single tracking in a recording contiguration The Beam Path and Channel Assignment panel displays the track configuration of the track currently selected in the List of Tracks panel highlighted in blue or gray The settings for this panel are performed separately for each track in the same way as for single tracking To do this select the track to be configured from the List of Tracks panel see the following description of the List of Tracks panel 4 60 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 2 List of Tracks panel List of Tracks In the List of Tracks panel the available tracks are Suden tes aier Ste Frame Frame Fast displayed with names activated channels and laser 1 lines FITC ChL1 ChL2 488 532 H Track ChL1 639 The sequence of tracks to be processed can be l changed for the scan procedure The Add
133. two vertical scrollbars for the setting of the image viewing angle e Select the required 3D display mode with a click of the mouse The additional function elements of the 3D display mode have the following meaning Profile Dist Profile Dist slider Setting of the distance of profiles and the mesh value of the grid Fill Level Fill Level slider Used to push through a color LUT Look Up Table e g if the Rainbow palette is used in the Profiles Filled display mode In combination with the Volume button the filling of the flood function level of the topography can be varied for volume measurements see the Measurement functions paragraph 03 06 B 45 0019 e 4 285 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan The image viewing angle is set as follows Setting directly in the image e Click in the image and hold down the mouse button The perspective is changed by moving the mouse button in horizontal or vertical direction Setting via scrollbars e Move the amp horizontal scrollbar to rotate the image around the vertical axis The rotation angle is displayed in the yellow display box e Move the 3 left vertical scrollbar to rotate the image around the horizontal axis The rotation angle is displayed in the yellow display box e Moving the J right vertical scrollbar enables you to expand the image in height or to compress it while the Z range between 10 and 100 of the X range is scaled
134. um Scale 25 65 pm ip SS y ChL1 T1 LChL2 T1 a uri 9 uri Optimal Interval 3 93 pm Optimal Pinhole Diameter Fig 4 63 Optical Slice window Tabs Z Sectioning Mark First Last Hyperfine Z Sectioning First Mid Last Refr Corr X Y Z 1 1 1 Auto Z Corr 4 80 Tab for setting of Number of Slices Interval and Current Slice via slider arrow button Tab for determination of the Z value for the first and last XY image of the stack combined with manual focusing or Stage control Tab for production of a Z Stack using the optional Piezo focus for objectives Scanning Display of the beginning first XY image of the stack Scanning Display of the center XY image in the center of the stack Scanning Display of the end last XY image of the stack Considers the different refractive index between the immersion medium of the objective n and the embedding medium of the specimen n which can be set between 0 5 and 3 via the slider arrow buttons n Ratio n Clicking on this button will set the Z interval in such a way that the voxel has identical dimensions in the X Y and Z directions cube This function permits the set values of the scan parameters Detector Gain Ampl Offset and Ampl Gain as measure for the brightness level to be varied between two freely selectable slices of a stack to be recorded During the scan procedure the interim values of these three parameters are a
135. unit is a single Time Series iy Block in line frame or Z stack mode In each block Limi Cal the user can define a configuration for the data acquisition single or multi track the number of iC T R m H Mee To images and the time interval delay between Humber of Seana prie 0 a oy Images 2 The user can activate an optional autofocus 729 00 Rerek Pon 1 Sn kI function before each block pre program a time pioni I Tigges Start Paure Plasume Trigger Trigger interval betore each block from the beginning of eee the previous block to the beginning of the current insane Daia Baie ieAlnana ditahase VIS EVE itinaas databada VIG EVS Mb block and or execute a bleach track with Tmp imaga Folder C arbitrarily specified bleach ROl s laser line s and Operon be Latiton 4 ihg power Open DE The autofocus function can be executed using a specified single track configuration or with the configuration used for imaging using the first channel of the first track Fig 4 174 Multiple Time Series main dialog box One can define the z offset the distance in the z direction from the position where the autofocus finds the reference feature in focus position of maximum intensity e g position of the cover slip reflection in the reflected light configuration to the position which is moved into focus plane when the image acquisition begins e g into the tissue The image acquisition is don
136. 0 11 Time secon ds Chl gt e Ch3 Ch Ready 151 x 87 x 8 3 channels 8 bit Display Zoom 1 Palette No Palette Fig 4 268 Image Display window Mean ROI display for time series in single plane 03 06 B 45 0019 e 4 265 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Pasi SCT eo Mia Tia M i chr be ee map tore Degg Tomes 1 Peete fie Peete interest 191 0210 Cb 1 Fig 4 269 Image Display window Mean ROI display for time series of Z Stack 4 13 22 Display 2 5 D This function allows to display the two dimensional intensity distribution of an image in an pseudo 3D mode show the intensity values in profile grid or filled mode show separate distribution for each channel in a multi channel image IB The 2 5 D button can also be used online during scanning e Click on the 2 5 D button The Pseudo 3D toolbar is displayed The settings of Slice apply The settings of Chan Zoom Contr and Palette are not applied Click on 2 5 D will display the Pseudo 3D toolbar Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed The viewing plane of the Image Display window can be rotated tilted either directly with the mouse or by the scroll bars on the right hand side and the bottom of the Image Display window 4 13 22 1 Direct Setting in the Image e C
137. 0 3 Beam Splitters MBS NT 80 20 D551 None DESA Miror NDD MBS None Wavelength 458 nm 4 1 2 Filters Che LP 385 Pinhole Ch2 896 um Total 1 471 MByte Selected 145 497 kByte Current 145 497 kBute Clipboard 337 063 kByte Filter Falter Delete Fig 4 10 Image Database window Form display mode In the Options menu in the function Settings it is possible to define the start mode of the image database Form Gallery Table the Recordset displayed first last middle and the parameters shown The number of the image currently displayed from a set of images is indicated in the Recordset text box e From the image database you can display images using the recording slider or in one of the following ways show the next image 4 show the previous image gt I show the last image of the image database 14 show the first image of the image database 03 06 B 45 0019 e 4 21 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan The currently selected image Is displayed as thumbnail in the Image Display window on the right In the case of Z Stacks or time series the Slice slider appears on the screen beside the Image Display window e You can browse through the series by dragging the Slice slider using the mouse e Click on the Load button to open the selected image IB A double click on the Image Display window of the database will also load the selected image
138. 0 pm Z e 7 Ha HRZ step um Focus step um pos F gt mm stage Position W Manual T step pm Po a Curent Fos x 1988 00 y 1988 00 Marks zen l OOO y O00 z O 000 Mark Pos Move To Remove Remove All HAZ Zero Move To Start HI g Stop m Y O12 Pixel Tile Numbers i A Tile Size s 512 Pixel Frame Size 1 512 Piel m 921 28 pm Y 512 Fisel Y 921 26 pm Fig 4 101 Stage and Focus Control window LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 9 Stage The following software description applies to systems which are equipped with a motorized stage This window enables you to activate both the motor focus and the scanning stage The Focus Position and Stage Position panels include the function keys for the performance of defined moves and the display of the current Z and X Y positions By use of an LSM 5 LIVE scanhead on an Jer 200 M sideport system care should be taken when moving the motorized XY scanning Stage to the maximum positions so that fingers are not bruised between scan head and stage 4 5 9 1 Open Close the Stage and Focus Control Window e Click on the Stage button in the Acquire subordinate toolbar of the Main menu The Stage and Focus Control window appears on the screen e Click on the Close button in the Stage and Focus Control window to close this window The following functions are available on the right h
139. 0 x 10 mm2 The scanning field size can be freely selected between 4 x 1 and 512 x 2048 pixels It is possible to carry out fastest XY scans without having to rotate the specimen itself under laser radiation load Selection of the specimen detail of interest for zooming Is fast and convenient and the zoomed image is automatically centered This saves the job of specimen centration with the microscope stage Using a bi directional scanning facility will double the scanning rate to 120 frames sec at 512 x 512 pixels if two different lasing wavelengths are used for the scanning two fluorochrome dyes can be viewed and documented in a quasi simultaneous mode This will absolutely prevent bleeding 03 06 B 45 0019 e 3 5 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 LIVE Carl Zeiss Performance Features of the LSM 5 LIVE LSM 5 LIVE DuoScan 3 4 2 Microscope Equipment of the LSM 5 LIVE System The LSM 5 LIVE system is equipped either with the inverted Axiovert 200 M SP microscope or with the upright Axio Imager Z1 Axioskop 2 FS MoT or Axio Imager M1 microscopes Referring to the delivered operating manual Axiovert 200 M only differences to this manual will be explained 1 Stand a The motorized objective nosepiece 5x H DIC is firmly fixed to the stand where no operating elements can be found for the nosepiece Operation will be done LSM 5 software controlled The Restriction of revolver height to protect the objectives when changi
140. 1 4 13 2 4 13 3 4 13 3 1 4 13 3 2 4 13 3 3 4 13 3 4 4 13 4 4 13 5 4 13 6 4 13 6 1 4 13 6 2 4 13 4 13 7 1 4 13 7 2 4 13 7 3 4 13 7 4 4 13 8 4 13 8 1 4 13 8 2 4 13 8 3 4 13 9 4 13 10 4 13 11 4 13 12 4 13 13 4 13 14 4 13 15 4 13 16 4 13 17 4 13 17 1 4 13 17 2 4 13 17 3 4 13 18 4 13 19 4 13 20 4 13 20 1 4 13 20 2 4 6 OPERATION LSM 5 LIVE Purpose LSM 5 LIVE DuoScan Close All Image Display WINQOWS sirsie snmerecenctaledodiuci cid duseetscaderseteroiasncuenicnesestadocendaens 4 221 Scan and System Information 00 c cece ee cece cece ee eeececee eee eeeeeeeeee ee eeeceeseeeeeeeeeeesaeeeeeeeaaneeeeees 4 222 Help MONU anena E E EEA NAN AAAA AEE EE 4 223 I o S E E E A E A E EAE E AE A T 4 223 ADOG e E A E E E eit tae eect eects 4 223 PRAP G UOO rae E E E E E EOE EE 4 223 Display and Analysis of Images cccccceeeeeseeeeeeeceeeseeeeeseeaeseeeesaeaaseeeeseoeeneeessaoaes 4 224 Structure of the Image Display WINdOW siseecicers shswesddarseseveacnne sivecbleevoncterddnocssdetumeuSeneeeas 4 224 DYES 0 Fe ANI 010 SSS 4 225 DOCG CAIN waversea ras E E E E 4 226 Assigning Another Color to a CHANING ivaccaeineinnsutanentevantencentcesagtnlondumnoudaunnivamencieatenia 4 227 Switching a Channel of a Multi Channel Image off OF ON esssnseessiiiisessrrrieeesrrrieee 4 227 Switching to Monochrome Image Display ccccccceccccceeeceeee ee eeeeeeeeeeeeeeaeeeeeeseeeees 4 227 Deinin
141. 1 No Fit 2 Plane The topography is tilted in such a way that the mean deviation value plane is calculated 3 Manu Tilt A 3Point Tilt 5 Sphere fit correction A spherical form is eliminated determination of micro roughness on spherical surfaces can be performed 6 Cylinder fit correction A cylinder form is eliminated determination of micro roughness on cylindrical surfaces can be performed I You can display the exact values of the Cylinder Sphere fit by opening a context menu in the Image Display box with a click of the right mouse button and selecting the Show processing parameter function Manu Tilt button Clicking on the Manu Tilt button opens the Manual Tilt window Fig 4 283 for manual tilt correction In the manual tilt window three types of arrow buttons are present One arrow changes pitch and yaw in 0 001 steps Two arrow changes pitch and yaw in 0 01 steps Three arrow changes pitch and yaw in 0 1 steps e Click on the Inverse button for the inverse display of the topography Clicking again will reset the normal display e Correct the tilt via the Fit or Manu Tilt functions Manual Tilt X I gt gt gt gt gt 0 000 Close cK gt gt gt gt o 000 Fig 4 283 Manual Tilt window 03 06 B 45 0019 e 4 283 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 24 5 Display Modes The three buttons in the
142. 1 20 1 14 Wise ga ET CE eea T E 1 21 1 14 1 Mounting and Dismounting Lamps TPMT and Switching Mirror esseessisessiieeesreeernee 1 21 1 14 2 Mounting and Dismounting the Scan HOAOS eirisi wacoctshoouteddaiasdvsinblaagh outed askahindGedadoedsbiunion 1 23 03 06 B 45 0019 e 1 1 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss General LSM 5 LIVE DuoScan 1 NOTES ON DEVICE SAFETY 1 1 General The LSM 5 LIVE laser scanning microscope including its original accessories and compatible accessories from other manufacturers may only be used for the purpose of microscopic techniques Laser Scanning Microscopes LSM are intended for high resolution imaging of biological or material samples whereby in contrast to wide field microscopy the specimen is illuminated raster tashion with a focused laser beam and the optical arrangement prevents light from out of focus regions of the specimen contributing to image formation IE Installation and commissioning of the LSM 5 LIVE system must be performed by authorized Carl Zeiss service staff The system should not be used prior to instruction by a Carl Zeiss representative A The manufacturer will not assume liability for any malfunction or damage caused by any thing other than the intended use of the LSM 5 LIVE or individual modules or parts of it nor by any repair or other service operation performed or attempted by persons other than duly authorized service staff Any such action will invalidate any claim
143. 1 to the image sequence Output If the grey value of the voxel is 0 then the voxel value of the Output image sequence is taken over If one or both input sequences are multichannel sequences any number or combination can be selected The number of selected channels for Input 2 must be 1 or the same as for Input 2 They will be combined from left to right Parameters Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence 6 40 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Scrap This function deletes or selects objects in a specified size range Scrap nu fF far 2 gt Output 2 Hew Minimum olume bo H H 0 153622 0 micrometer Masimum olume hooo HT H 0 153624 0 micrometer Select E Cancel Apply Fig 6 31 The operation deletes or selects objects on the basis of their total volume in voxels Objects with a volume within the range MinVolume to MaxVolume are effected To delete objects outside the range the parameter Select must be active If the parameter is not activated objects outside the defined volume range are deleted Parameters Input Input image sequence Output Output Image sequence MinVolume Minimum object size MaxVolume Maximum object size Select O Select the objects outside the size range 1 Select the regions within the size range 03 06 B 45 0019 e 6 41 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions L
144. 2 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss e Click on the desired configuration The selected configuration is shown in the first line of the Configurations list box e g DAPI e Click on the Apply button The program loads those parameters of the selected Channel Mode Configuration which have been activated in the Options menu under Settings Recording Configuration see Settings Function page 4 200 The Channel Mode Configurations window is automatically closed cr The optical diagram of the configuration selected appears on the Beam Path and Channel Assignment panel The entire recording configuration has been activated for the scanning procedure b Store a Channel Mode Configuration A newly created or changed recording configuration can be stored under a new name as follows e Click on the Config button the Channel Mode Configurations window appears on the screen e Enter the desired name in the first line of the Configurations list box e Click on the Store button e Close the window by clicking on Close During storage via the Config button all the data of Beam Path and Channel Assignment and the Detector Gain Ampl Offset Ampl Gain and Data Depth 8 12 Bit scan parameters of all the defined tracks multitracking are stored Furthermore the used objective the Frame Size Zoom Rotation amp Offset and Scan Direction parameters are stored c Delete a Channel Mode
145. 256 or scan Direction Number 7 4096 gray values e Select the Unidirectional or Bi directional Scan Direction Fig 4 52 Pixel Depth Scan Direction amp Unidirectional The laser scans in one Scan Average panel direction only then moves back with beam blanked and scans the next line Bi directional The laser also scans when moving backwards i e the Scan Time is halved Pixel Depth Scan Direction amp Scan Average E Unidirectional scan is always optimal regarding image quality For possible artefacts caused by Data Depth 8 Bit 260 Saa one i bidirectional scan partially caused by fluoro me Madj Mean E chromes behaviour jitter or flicker the Scan Corr Number 1 z Y correction slider is available Move slider to either side in continuous scan mode till images are stable os The Auto button sets the right correction Ee SS E ani l automatically Fig 4 53 Bidirectional Scan amp Scan Correction 4 72 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Example Intensity Intensity 250 250 200 200 100 100 50 4 a 3 4 5 6 Time s nc 4 RO Z Fig 4 54 Example for Scan Correction Intensity fluctuations at the end of the frame left image are fully compensated by the Auto button right image Average e Select the Line or Frame mode for averaging e Select the desired scan average method Mea
146. 4 178 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan Preparations for using the Calibration Slides 03 06 Follow the steps shown in the macro Sample and Configuration Setup window Use the arrows or the drop down menu in order to select one track for each available laser line no need to select several tracks for the same laser line start with a track for the lowest wavelength laser i e typically the 405 nm laser line Select an area of the bead sample that provides as many beads as possible at least 4 Tit into the green rectangle In Fig 4 176 the Basic Calibration has been selected Check box Basic Calibration Requires selecting preferred objective lens therefore some more tracks are available Use the Laser Power as a first option in order to set the intensity second option is Integration Time In order to start stop the scan just select deselect the check box Continuous Scan Focus on the beads Defocus with the Collimator slider until the beads become line shaped Focus with the microscope focus drive to get the vertical lines as thin as possible Fig 4 177 Now use the Collimator slider to get a round spot out of the line Fig 4 178 Repeat these steps for a track with the 488 nm laser line After one track for each laser line has been adjusted click Next B 45 0019 e OPERATION Macro Menu Carl Zeiss sample and Configuration Setup ocus on the sample Make sure that at least
147. 4 6 7 1 Open Close the Filter Window e Click on the Filter button in the Process subordinate toolbar of the Main menu ae Se This opens the Filter window fete ned ts Oa ae 2 4 2 e Click on the Close button to quit the Filter oleae E ee window Preview O T Preview 4 6 7 2 Image Panel In the Image panel the image or channel to be Fig 4 117 processed is selected Filter window The currently selected image is displayed in the image selection box Proceed as follows to select an image via the image selection box e Click on the arrow button The image selection box is opened and all the currently loaded images are displayed in a minimized form e Click on the required image which will then appear in the display box of the image selection box and will be available for filtering L Youcan also use the Click into window button to select the image e Open the Channel selection box with a click on the arrow button and select the channel to be processed 4 6 7 3 Filter List and Edit Panel In the Filter List panel the filters and the matrix size Kernel Size are selected The matrix of the selected filter and the set filter parameters Factor Divisor and Offset are displayed in the Edit panel 03 06 B 45 0019 e 4 129 OPERATION Process Menu Carl Zeiss User defined Kemel Size 3x3 aS Be ee a E Se S Lae VT ee ew Eo sg ee ea ia Ea Offset cr EESE a
148. 4 Select Save As This function allows to save the image s of the Image Display window into an Image Database by showing a dialogue use the defaults as defined in the Settings function with the Save tab Click on the Save As button displays the Save Image and Parameter As window Changes will be effective on closing this dialogue In the Options menu in the function Settings with the tab Save default parameters such as Name Description and Notes can be set e Click on the Save button The Save Image and Parameter As window appears e Enter text for the image name description notes or change the user name e Select the Image Database from the list of databases MDB or e Open other Image Databases by selecting Open MDB or e Create new Image Databases by selecting New MDB 4 13 15 Display xy This function allows to display a single image in frame mode display multi channel images in superimposed mode The settings of Chan Zoom Slice Overlay Contr and Palette are applied Click on xy will be immediately effective 4 244 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 16 Display Split xy This function allows to display the individual channels of a multi channel image as well as the superimposed image The settings of Chan Zoom Slice Overlay Contr and Palette apply Click on Split xy will be immediately effective OLX
149. 5 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 23 5 Series Render Series FS Projection The Series button opens the Render Series on PANETER window This window allows settings for the axis C Turm around Y to be used for rotation of the 3D reconstructed Start and End C Position List Appl Images Time senes Number of Views 10 5 1 4 8 18 2 64 os e Click on the Series button to open the Render Difference Angle ra 5 a5 18 3 Panorama Series window First Angle fo j 2 Save e Select the requested projection mode by 3 clicking on the relevant option button under Mode Ws e Depending on the activated mode directly set the parameters for animation in the Render Series window and the position of the 3D interpolate image in the Image Display window zoom rotation axes rendering parameters e Click on Apply to start the animation The animation is performed in a separate Image i Fig 4 276 Render Series window Display window which permits the animation to e g Turn around X mode be saved afterwards 1 Turn around X and Turn around Y mode In this mode the image is turned around the X axis or the Y axis exclusively The values for Number of Views Difference Angle and First Angle can be selected accordingly see Projection page 4 155 03 06 B 45 0019 e 4 275 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of I
150. 6 Antiglare screens for Axiovert 200M left and Axio Imager and Axioskop 2 FS right gt Use these screens for improved working ergonomy 03 06 B 45 0019 e 1 11 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss Protection against white light LSM 5 LIVE DuoScan 1 5 Protection against white light emitted around the objective and sample by an X cite fluorescence illumination Follow the guidelines in the X Cite manual provided by EXFO Inc Don t use a neutral beamsplitter e g NT80 20 position in LSM DuoScan or LSM 5 DUO systems in the P amp C modules while looking through the eyepieces Strong white light might project into your eyes In case such strong light is projected into your eye the natural lid closure effect will protect you from any damage Nonetheless such exposure is unpleasant and should absolutely be avoided 1 6 Notes on Setting up the Microscope System Installation and commissioning of the LSM 5 LIVE system must be performed by authorized Carl Zeiss service staff The system should not be used prior to instruction by a Carl Zeiss repre sentative The LSM 5 LIVE laser scanning microscope is delivered in several crates A The LSM 5 LIVE must be set up so as to ensure that the minimum clearance between the wall and the rear of the system is no less than 0 5 m This clearance is needed for adjustment and maintenance operations Do not set up the unit in the proximity of heat sources such as radiators or
151. 65 0 819763 0 258096 16 188839 0 061771 0 303808 EREE Copy Table Time Intensity Ch2 T1 2 s Save Table 0 0000 i 0 0464 0 0625 0 1409 0 1250 0 1957 0 1876 0 2253 0 2501 0 2776 0 3126 0 3063 i 0 3751 0 3223 Info Nn Az N 35PR zl Ready 98 x 80 x 1 483 1 channel 12 bit Reconstruction Palette Palette from Image File Fig 4 142 Image window displaying the analysis of FRAP data using a double exponential fit The raw data of the experiment can be exported for further analysis using the Mean ROI display mode and within this dialogue the table display of the results Note that this modeling is also a very basic approach to your experiment It offers a first hint on the possible presence of two mobile fractions of the labeled protein within the cell or cell compartment examined Please refer to relevant scientific literature or the website of the EAMNET htto Awww embl de eamnet for further information on how to set up and perform FRAP experiments A schematic curve marking the data points that are calculated performing the Kinetic Analysis is shown in Fig 4 143 Please note that the naming of the data points is not consistent with the information on the website 4 150 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss delta i tia fraction l1 mobile fraction Amplitude Acie 2 ee i a PE cheered iIi Fig 4 143 Schematic FRAP curve with marks at th
152. AFETY CONTENTS Page 1 NOTES ON DEVICE SAFETY ccsicemnana sss cemeteries sce cect aes 1 2 1 1 GOON AI se here tionct en occur netecaen te eine ante aa sorn reo ica ritce Saoegi Norn ate ernie manger ache cine 1 2 1 2 sVAVecl gall ae les ACM AUK elAnarsid o aie kes 6 S elenenn ametne metre nee are enna nr mre mate mn eren en eee eee ae see om 1 3 1 3 REIED D a Sane men emer re tance E PE A E E E EEE ee A ee E eee 1 10 1 4 Protection against diffuse laser excitation emitted around the objective and sample 1 12 1 5 Protection against white light emitted around the objective and sample by an X cite Haorescence MNT AE ONN etn Gest aaa eta A 1 13 1 6 Notes on Setting up the Microscope System cccccccceccccceseeececeseeeeeeeseeeeeeeeeeeeseeeeneeeeenaes 1 13 1 7 POwWeErREGUTEMEN S renee tee hoses chs a a e ilaitliainudeuela din Mars tralusaieeades sided 1 15 1 8 Notes on Handling the Laser COMPONENMS cccccceeeeccce see eeeeeseeeeeeseeeeseeseeeeseeeaneeeeanaes 1 16 1 9 Notes on Handling HBO and X Cite Light SOUPCES 20 0 cece cccce cece ccee ce eceeeseeeeeeeaneeeeeenens 1 18 1 10 PIA SICA DIMENS ON ra aaa a Pethetaiaie thane Suki 1 18 1 11 Environmental Regule meni eoira ioa iT ine a a tose atiaie 1 18 1 12 Notes on Handling the Computer and Data Media ssssnnssssiiusssriussirnesrrrrerrrrrerrrrrens 1 19 1 13 Notes on Care Maintenance and Service 22 0 0 ccccccccccccececcececcecuccucecceceecucecaeseeaucutaeeevaneess
153. AN r A 6 37 OT a E AS adenine 6 37 Ol a A veda nates 6 38 Camera color adjustment 000cccee 4 220 Camera detection ccccceccecccceeeeeeeeeeeees 4 66 Carl Zeiss vision image sequences 0 6 12 03 06 B 45 0019 e List of Key Words Carl Zeiss GANG aana a a aa 4 75 4 226 Add reMOVe ca shnitaitutatt ices atmatiendonieds 6 5 6 15 DEET rana an 6 16 Channel assignment 4 55 4 60 4 316 4 322 Channel mode configuration 6068 4 62 COSTA aeS 6 32 Collimator adjustment ccceecceee eee 4 218 CONOR pal EU arca EE 4 237 Configuration control s s 4 51 CONTAS ste piace tanec cnlei uel conltnes 4 132 4 234 MANGE CONTEST seseris kinoen 6 19 BIANCO i inbiatudburteiedstehyid od 6 23 Clo 0 Ree ener ee meen ree eee eer ey 4 25 4 243 Content of display window 0668 6 14 Copy full resolution goscctvacetuoustatecicacmonsecss 4 146 Copy window CONteEntS n 4 145 Gi 0 6 meo 4 242 Uh ee ee eae tee ee ae ee ee 4 250 D Database e ncdu nce cocina tatne eine andandeadtastcaewad 4 18 Belete TUN CUON a cece urecdirenanedtd cateeness 4 28 Export of IMAGOS n 4 33 EEr UNC CUO een 4 26 FOrmM MOQE scat dnedev ice tusdexsecneilucatibeaxwant 4 21 Galery TOGO niinen ane 4 23 Import of IMageS as eeneiieenniineeen 4 32 Load fUNCTION cccececcecceeeeees 4 24 4 25 Multi PriNt ieaie eee ene es ere nr eat ae er 4 34 INOW A ET E cect eee sada esate t
154. ATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 23 3 Surface Rendering The surface rendering generates surface rendered 3D reconstructed images The elements for image display zoom 3D rotation home coordinate system scale frame and animation function are identical to those of the transparency projection and are operated in the same way The surface projections Basic and Advanced are perspective type reconstructions of the surface and differ in the fact that the calculation of the 3D information is based on voxels or triangles The Advanced projection permits the View angle to be varied in order to enhance the perspective impression It is also possible to select between fast and precise calculation via the Precise Fast slider at the bottom right in the 3D toolbar Of course the precise calculation method is more time consuming The Full resolution projection is based on a high precision calculation method for 3D information on the basis of triangles with maximum resolution L Depending on the hardware configuration it can take several seconds until the surface projection is refreshed on the screen 03 06 B 45 0019 e 4 273 OPERATION Display and Analysis of Images Carl Zeiss 3D Rendering E Shading Model List Distance i i j D Azimuth 30 0 Elongation fo aq Remove Material Channel E UUl Che Cha Use channel colors
155. Axiovert 200 M SP 100 nm Axioskop 2 FS MOT 10 nm Axio Imager Z1 Piezo driven single objective drive Max travel 250 um resolution 5 10 nm In the unlikely case of extreme fluctuations of the external power net or electromagnetical radiation the piezo crystal will vary and disturbance in the image Is visible Note that this is not a defect and the piezo drive will not be damaged B 45 0019 e 2 11 LSM 5 LIVE SETUP REQUIREMENTS Carl Zeiss Scanning Module Laser Module LIVE LSM 5 LIVE 2 14 Scanning Module LSM 5 LIVE Scanners 2 individually driven galvanometric scanners Scanning speed Up to 120 frames sec 512 x 512 pixels Field resolution Max 512 x 2048 pixels optional adjustable for each axis Field of view 10 x 10 mm with a 1 25x objective Zoom 0 5x 2x continuous control Channels Up to 2 channels simultaneously Optional transmitted light detection mode Dynamic range 12 bit DAC for each detection channel Apertures 1 or 2 individual variable aperture Computer controlled automatic alignment 2 15 Laser Module LIVE Single mode polarization preserving Tiber Laser beam attenuation AOTF for all lasers Diode laser 405 nm 50 mW Diode laser 440 nm 16 mW OPSS laser 488 nm 100 mW DPSS laser 532 nm 75 mW Diode laser 635 nm 35 mW In the unlikely case of extreme fluctuations of the external power net the laser diode will switch off or the laser power might vary Note that this is not a defect a
156. Both shutters are closed automatically when switching to LSM mode Shutter Opens and closes the shutter in the reflected light path independent of the status of the internal lamp shutter and the attenuation Intensity Sets the light via input box or slider Lamp Hours Indicates the total time the lamp has been in use 4 5 2 4 Working in LSM Mode Switchover to the scanning mode is then required e Click on the LSM button in the Acquire subordinate toolbar 4 5 2 5 Microscope Control for Axioskop 2 FS MOT For setting the Axioskop 2 FS MOT proceed in the same way as with Axio Imager Z1 and Axiovert 200 M Since the Axioskop 2 FS MOT is not motorized except the external Z drive and the tube all microscope settings have to be made manually Especially the change of objectives is made manually The used objective must be set in the Scan Control window 4 50 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 3 Configuration Control The setting of the beam path for the scanning procedure i e the definition of channels PMT photomultiplier and tracks and the setting of the attenuators of the various laser lines is performed in the Configuration Control window L gt A track is a set of parameters for the detection channels and for illumination wavelength and intensity scanned simultaneously and identified and handled by the system with one name The Configuration Control window has
157. CE SAFETY Power Requirements LSM 5 LIVE Carl Zeiss LSM 5 LIVE DuoScan 1 7 Power Requirements The LSM 5 LIVE comes with a mains power supply cord and plug either CEE red 3 N PE 400 230 V 16 A or NEMA L 14 30P 2 N Ground 120 240 V 30 A and with the matching mains socket outlet A ground wire AWG10 green yellow is supplied because it is necessary to ground the system The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter A suitable grounding point must be installed in the room For systems 220 240 V AC equipped with X Cite 120 the mains socket outlet must be equipped with a fuse having minimum tripping characteristic C according to IEC EN 60898 Line voltage Line frequency LSM incl VIS laser Max current Power Power Consumption Argon UV laser Line Voltage Max current Power consumption Class of protection Type of protection Overvoltage category Pollution degree 220 240 V AC 10 50 60 Hz 3 phases at 16A Phase 1 1 9 kVA max Phase 2 1 5 kVA max Phase 3 2 6 kVA max 5000 VA max 208 240 V AC 10 50 60 Hz 1 phase at 63 A Note For Line Voltage 220 V the con nector and power plug are rated for 63 Amps However wiring and fuse should be rated for 32 Amps 7000 VA max B 45 0019 e 100 125 V AC 10 50 60 Hz 2 phases at 25 A Phase 1 3 2 kVA max Phase 2 2 8 kVA max 5000 VA max 208
158. Carl Zeiss 4 6 12 lon Concentration E lon Loncentration The use of this function option ra permits the calibration of ion lon Core concentrations in physiological experiments real Suge arang bu Meh 4 6 12 1 Open Close the lon Concentration Window e Click on the lon Conc button in the Process subordinate toolbar of the main menu Raons Dye ee ee mat A Fimn Shon This opens the lon Concentration window a Fin Rue P T Equation z Ed Si e Click on the Close button Tieton ny a Ini Ran 0 10 Eck into ence T inyi Fria 200 ima ja 0 Dick rig vanda Min Concentration 0 00 S Max Cercentiion 200000 Fig 4 135 lon Concentration window 4 6 12 2 Function Description lon Conc button Activates the lon Concentration window Source window Selects input of images to be processed Destination window Select output and pixel depth of processed image Calibration window Sets the six different calibration options according to the dyes used single wavelength ratiometric and required method Show Curve button Shows resulting calibration curve Image scaling window Sets min and max concentration Preview window Activates Preview function 03 06 B 45 0019 e 4 141 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 12 3 Single Wavelength Dyes Offline Calibration e Subtract background autofluorescence image from raw images to obtain e Perform equati
159. Click on Close e Click on e Click on 03 06 the Objective button and select the objective by clicking on it the Reflector button and select the None B 45 0019 e Switch HBO 100 power supply Stage fine motion control X Stage fine motion control Y OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Microscope settings on Axiovert for reflected light observation Epi fluorescence e Turn on the HBO 100 power supply switch 4 32 1 e Click on the Reflected Light button and set the shutter in the Open position e Click on the Reflector button and select the desired filter set by clicking on it e The filter is automatically moved into the beam path to enable observation in epi fluorescence e Click on the Tube Lens button and select the tube lens e Click on the Objective button and select the objective SP If the X Cite 120 fiber coupled lamp for Shutter reflected light illumination is used a Close further dialog is opened when clicking Memel uui Ue the Reflected light button The lamp pJ N as internal shutter and the attenuation function can be controlled via the LSM Software The lamp has to be switched on first using the main switch on the Remote On Fig 4 33 X Cite 120 control panel lamp housing Remote Enables the attenuation control via LSM Software This is also enabled when clicking the ON button ON Opens the shutter in the reflected light path and the internal shutter of the lamp
160. Click on the Scan New Images button in the LSM 5 LIVE Switchboard window Clicking on this button activates the complete LSM hardware on line mode e Click on the Start button in the LSM 5 LIVE Switchboard window The LSM 5 LIVE Main menu appears on the screen Use of this mode requires to be thoroughly familiar with the exact microscope procedures and interrelations PET Whe aae P Sey mre tf 1 Main menu pull down a ee ES a i fe Tc er Tl F my si ee ER l F a Fy i oe l i a a a f bite 1 1 rae 1 LES To TF ta 5 J 1 j i bA aa Pre i Li Fig j m hy Efi ing F m Ari bef ETS Pi PE zij i at PPN E F j Pei Tre l 2 lt Main menu toolbar pe ay lt Subordinate toolbar Fig 4 Main menu 4 03 06 Creating a database for acquired images e Click on the File button in the Main menu toolbar The File subordinate toolbar appears in the Main menu Maintain F Options TE O Fig 5 Main menu with File subordinate toolbar Mi e Click on the New button in the File subordinate toolbar Neve The Create New Database window appears e Select drive C or D from pull down menu e Create a new directory if needed Create New Database Create New Database Create in E My Computer EE ee Create in le D cs Removable Disk E amp IF IG File name Create File name Create Create tupe Database Files m
161. Click on the Start button in the LSM 5 LIVE Switchboard window The LSM 5 LIVE Main menu appears on the screen The Acquire button is active automatically and the submenus selectable in it are shown in the second bottom toolbar Main menu Main menu Subordinate pull down toolbar toolbar Fig 4 5 LSM 5 LIVE Main menu File button Open save import and export of image data Printing individual or several images on one page Ending Exit the Main menu I ene Acquire button Calling up and setting the necessary operating parameters During the preparation for and execution of laser scan image acquisition this menu item is used as the working dialog between the computer and the microscope i en Process button Used for processing of acquired images E 2D View 3D View button Used for three dimensional reconstruction 03 06 B 45 0019 e 4 15 OPERATION LSM 5 LIVE Carl Zeiss Main Menu LSM 5 LIVE DuoScan B Macro Macro button Makes it possible for the user to store frequently used processes Macro recorder and to run them automatically Macro play It is possible to write new macros or to edit existing ones Options Options button For custom configuration of software and hardware options This menu item enables access to the coloring table In the Settings for User window you can specify essential operating modes and informative help organized by tabs which have an effect on the user interf
162. Color Number of Pixels 1300 H x 1030 V 1 3 Mega pixel Chip size 8 7 mm x 6 9 mm equivalent to 2 3 Spectral range Limited by IR barrier filter BG40 about 400 nm to 700 nm Selectable Resolution by Binning or Microscanning H x V Acquisition Time s 20 ms exposure 432 x 342 0 07 Color interpolation 1300 x 1030 0 2 Color interpolation 1300 x 1030 0 7 full resolution for color channels 2600 x 2060 3900 x 3090 Dynamic Range Typical 2000 1 9 e readout noise Integration Time 1 ms to several minutes Cooling One stage Peltier cooling Optical Interface C Mount Size about 11 cm x 8 cm x 6 5 cm 2 3 x 3 2 x 2 6 Registration GS CE cUL Power Supply 12 V DC 1 A 230 V 110 V autodetecting 03 06 B 45 0019 e 7 15 ANNEX LSM 5 LIVE Carl Zeiss AxioCam High Resolution Digital Cameras LSM 5 LIVE DuoScan 7 8 4 Microscope Camera Port Adapters for the AxioCam Adapter Video V200 C 2 3 0 63x at frontport Axiovert 200 M Cat No O00000 1071 171 This adapter is needed for attachment of the high resolution AxioCam microscope cameras on the Axiovert 200 M Adapter Video 60 C 2 3 0 63x Cat No 000000 1069 414 This adapter is needed for attachment of the high resolution AxioCam microscope cameras on the Axio Imager Z1 and Axioskop 2 FS MOT For an additional documentation port to be connected to the camera port of the tubes with interface 60 Double video adapter Cat No 000000 1058 640 For connection to interfa
163. Color selection box Source 2 selection box with Color selection box Selection of the second channel to be selected via the selection box assignment of a defined color via the Color selection box Mask selection box with Color selection box Selection of RGB or Overlay display of the mask assignment of a defined color via the Color selection box Allows the selection of ROIs in the Histogram and the Image Stores ROIs and threshold settings B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Enhanced colocalisation Scatter diagram image display and data table are interactively linked 007 GPP WTP mi Ali iI T i S PO 1 ou AST Rpa Plesechy IOBA x DOM 3 channa 12 ba Fig 4 264 Image Display window Colocalization display Scatter region 3 Colocalizing pixels Ja scatter region 2 Pixels in channel 2 only Scatter region 4 Sub treshhold pixels background intensities Scatter region 1 Pixels in channel 1 only 0 500 1000 1500 2000 2500 3000 3500 4000 Intensity GFP 50 100 150 Absolute Frequency Fig 4 265 Scatter diagram and threshold with crosshair 03 06 B 45 0019 e 4 259 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Quantitative Parameters No of pixels in image ROI or scatter region Area relative area of image ROI or scatter region Mean intensities SD within imag
164. D Grey value standard deviation of all regions Densitometric Minimum VolIMinD Minimum grey value in the image sequence Densitometric Maximum VolMaxD Maximum grey value in the image sequence Densitometric 6 56 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Automatic Object Measurement Condition The measurement conditions are used to limit the objects to be evaluated e g only objects with defined minimum value All objects are tested against the defined conditions If the conditions are fulfilled the feature values are written to the data table dg Automatic Object Measurement Feature Operator Number List of conditions Remove All IAA tou oi Minimum Yolurne E voxel Cancel Apply Fig 6 39 To define the following parameter select the Condition tab sheet of the Automatic Object Measurement dialog window The list on the very left at the dialog shows all the measurement Features The second list provides the comparison Operators and the next Numbers to define a value This gives the possibility to compose an expression to test a feature value against a constant value The fields above the lists will show the composed selected string Clicking on the desired list entry does the selection The button with the gt gt Characters adds this string to the List of Conditions All lines of the List of conditions are combined with the AND expression automaticall
165. D topography are named with S The Copy button is displayed below the right hand side of the image This button permits the roughness parameters to be copied to the clipboard and imported to another program e g MS Word or MS Excel via the Paste function 3D Amplitude parameters Topography Roughness 03 06 B 45 0019 e 4 301 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Calculation of roughness parameters The following roughness parameters are calculated Mean height of all surface height values S N x Z x Y Nx Ny number of pixels in X or Y direction Me RS N N X l j l Arithmetic mean deviation of all surface height values RS a a D keyi es N x RS X Quadratic mean deviation of all surface height values RS ko o o 3 i R TE 2 es Kurtosis of the distribution of all surface height values Sky TE x RS E Z x y N N RSE Maximum peak height RS p Zaa m ha max C Maximum valley depth Sy Roi Shae Maximum roughness depth RS Peak to Valley PV SRI Z max min maximum height difference of the overall topography 4 302 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Classification of topography in 25 equal area elements rectangles in the 2D mode average roughness depth S po
166. Display button bar allow stacks to be displayed in the 2D Iso Lines or 3D display mode 1 2D modes The following 2D modes can be set PE Intensity button Display of projection of all intensities of the stack black and white Intensity display Height button Height coded color map with color bar in a separate parameter window E on the right hand side Gradient button Display of height gradient slope averaged pixelwise over all neighbors Gradient black and white display e Click on the 2D button in the Display button bar The 2D display mode selected last is activated At the same time an additional button bar is displayed beside the 2D button permitting selection of the required 2D display mode e Select the required 2D display mode with a click of the mouse 2 2D Iso Lines display mode Iso Lines are lines which connect points of equal height on the topography The following 2D Iso Lines display modes can be set Intensity button Intensity projection superimposed with colored iso lines lines of equal height Height button Height function with black Iso lines Black button White iso lines on a black background e Click on the Iso Lines button in the Display button bar The lso Lines display mode selected last is activated At the same time an additional button bar is displayed beside the Iso Lines button permitting selection of the required Iso Lines display mode Below the Measure b
167. DuoScan 3D View Menu Carl Zeiss 4 7 1 2 Source Panel In the Source panel the image source is selected The currently selected image is displayed in the display box of the image selection box Proceed as follows to select an image via the image selection box e Click on the arrow button The image selection box will be opened and all the currently loaded images are displayed in a minimized form e Click on the required image which will then be shown in the display field of the image selection box and be available for the following operation The Click into window button enables you to select the opened image directly e Click on the Click into window button first and then double click on the relevant Image Display window The selected image will then be shown in the display box of the image selection box Select the channels to be processed via the Channel selection box e Click on the arrow button to open the selection box Click on the required channel to activate it 4 7 1 3 Depth Coding Panel Depth Coding Transparency e On the Depth Coding panel you can set the ee 2u eal gree desired parameters Activate the Scale Bar Threshold oS check box ll if you want a color scale to be cane EE shown TEF aE Display M Scale Bar F Grey level Fig 4 146 Depth Coding tab 1 Depth Coding tab Mode Front View The image is viewed from the front above when this option is activated Mode Rear View The image is viewed from the re
168. E EA Bleach Parameter Panel ccccccceceecceceeeeeeeeseeeeeseaeeeeaeeteneeaeenes Excitation of Bleach Track Panel 0 cccceccecceeceececeeeeeeeeeeeeeaeenenes Start End a Bleaching Procedure cccccccceecccceeeececeeeeeeeeeeeeaeees UAC Gl EEEE EPN AAIE EET EEEE ETETETT ET PET EEEE Open Close the Stage and Focus Control Window 0 FOCUS POSITION Panel c cccceccecceeceeceeccecceeeeeeceeeeaeeeeeereneeenenteaetars B 45 0019 e LSM 5 LIVE LSM 5 LIVE DuoScan 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 9 5 4 5 9 4 4 5 10 4 6 4 6 1 4 6 1 1 4 6 1 2 4 6 1 3 4 6 1 4 4 6 1 5 4 6 2 4 6 2 1 4 6 2 2 4 6 5 4 6 3 1 4 6 5 2 4 6 4 4 6 4 1 4 6 4 2 4 6 4 3 4 6 5 4 6 5 1 4 6 5 2 4 6 6 4 6 4 6 7 1 4 6 7 2 4 6 7 3 4 6 7 4 4 6 8 4 6 9 4 6 9 1 4 6 9 2 4 6 9 3 4 6 9 4 4 6 10 4 6 10 1 4 6 10 2 4 6 10 3 4 6 10 4 4 6 11 4 6 11 1 4 6 11 2 4 6 11 3 4 6 12 4 6 12 1 4 6 12 2 03 06 OPERATION Purpose Carl Zeiss SOFO TON Pale orson o E N net abebicasteeassendaet 4 116 WE Salle ANG a a a a a a a a a a a a aa 4 118 VIS TV and LSM BUIOMSe 56st tae 5 be cottes atest ka date Sec tas nichols ort tod ehes dilea tits tess daptbeid oti 4 120 PROCESS VIC Ui tacaiacnececccenatinscaveruteeactenauaeapenedadancus cotaon ote cnaae sedeudeweat peter omecbdenteusmetmeecedaess 4 121 PANG Beco riitetie E A oad ecto ha tkeae angen eae hb cece 4 121 Open A ocete Add
169. Function option group in the Boolean dialog window must be selected This function can be used to combine binary masks or regions If one or both input sequences are multichannel sequences any number or combination can be selected The number of selected channels for Input 1 and Input 2 must be the same They will be combined from left to right Parameters Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence 6 38 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Boolean Not This function carries out a bit by bit negation of an image Boolean And Or xor Not Mask Input 1 i Alf 1 Output E Me Fig 6 29 The Not tab sheet of the Boolean dialog window must be selected If Input is a multichannel sequence any number or combination can be selected Parameters Input Input image sequence Output Resulting image sequence 03 06 B 45 0019 e 6 39 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan Boolean Mask This function masks a grey value image sequence Boolean And Orl sor Mot Mask Input 1 Moo Alf 1 Input 2 pooo A Output 3s sis Hew Fig 6 30 The Mask tab sheet of the Boolean dialog window must be selected This function modifies the Output image sequence depending on the mask image sequence used If the grey value in Input 2 is higher than 0 then the voxel values are copied from Input
170. HISTON KEV VVO G Sioe aa a r a a EN 7 17 03 06 B 45 0019 e 7 1 ANNEX LSM 5 LIVE Carl Zeiss Recommendations for excitation laser lines and LSM 5 LIVE DuoScan 7 ANNEX 7 1 Recommendations for Excitation Laser Lines and Emission Filters of Dyes Dye Laser line HFT Emission EM DAPI 405 gt 420 max at 461 Lucifer Yellow 440 or 488 gt 475 max at 536 EGFP gt 505 max at 507 516 FM 1 43 gt 505 max at 598 Alexa Fluor 488 gt 505 max at 520 DIO DIOC18 3 gt 505 max at 508 ine 405 405 488 4 4 4 4 4 uorecen a gt 650 max at 666 in 88 88 88 88 88 88 532 532 532 532 532 532 532 532 532 532 532 532 033 633 7 2 B 45 0019 e 03 06 LSM 5 LIVE ANNEX LSM 5 LIVE DuoScan Recommendations for excitation laser lines and Carl Zeiss Here you can note your specific combinations Example 7 2 Configurations Overview 7 2 1 LSM 5 LIVE 1 Channel Biomedical Configurations 1 channel FL VarioOne GB Laser lines 488 532 635 nm Emission LP 505 filters BP 495 525 SK channel 1 LP 550 BP 550 615 LP 650 SK BP 700 750 03 06 B 45 0019 e 7 3 ANNEX LSM 5 LIVE Carl Zeiss Configurations Overview LSM 5 LIVE DuoScan 7 2 2 LSM 5 LIVE 2 Channel Biomedical Configurations 2 channel FL VarioTwo VRGB Laser lines 405 440 488 532 635 nm Secondary Plate beam splitter mirror NFT 488 NFT 532 NFT 635 Emission LP 420 filters LP 505 chann
171. IVE DuoScan Contents Carl Zeiss CHAPTER 3 INTRODUCTION TO LASER SCANNING MICROSCOPY CONTENTS Page 3 INTRODUCTION TO LASER SCANNING MICROSCOPY cccceessseeeeeeeseeeeeeeeeeeeseeaeaes 3 2 3 1 Principle of Laser Scanning IWICLOSCODY ascent nnr riea A dances 3 2 3 2 Three Dimensional Presentations of LSM Image Stacks esssiinsesriiiuesrrirrrerrrrrrrerrrrren 3 3 3 3 Optical Diagram of the LSM 5 LIVE Schematic sgcutnaeadaced andi nedosdengdnebanadom tueteedetgeen eu teesidel 3 4 3 4 Pertormance Features Ol the ESM 5 LIVE aniis ee o R E a oa 3 5 3 4 1 Opicakand Mechanical ASPEC inanir o r E E E E E E 3 5 3 4 2 Microscope Equipment of the LSM 5 LIVE SySt M essneessiiessriresrrrresrrrrerrrrrerrrrreerrrrerrn 3 6 3 4 3 Computer Hardware and SOM Wale reani aE E E E E N AEA ETE 3 8 03 06 B 45 0019 e 3 1 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 LIVE Carl Zeiss Principle of Laser Scanning Microscopy LSM 5 LIVE DuoScan 3 INTRODUCTION TO LASER SCANNING MICROSCOPY 3 1 Principle of Laser Scanning Microscopy To yield information on their inner structure by conventional transmitted light microscopy specimens have to be very thin and translucent otherwise image definition will be poor In many cases it is a problem to satisty these requirements The essential considerations have led to trailblazing changes in conventional microscopy and supplied a successful solution to the above problem e Unlike the practice of
172. If nothing is marked the value of the variable above the text cursor is displayed Activates the Watch window in which values of variables and expressions can be displayed For this text is marked in the code window and dragged into the Watch window Variables can be modified in the Watch window In the left hand edge of the code window you will find an arrow beside the current command line A new current command line can be determined by moving the arrow via the mouse This makes it possible to skip command lines or to process command lines several times 4 172 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss 4 8 3 Overview of Available Macros Documentation files rtf doc of a AOTFfitlin lvo Autofocus vbo Bleach lvb CameraColor vb also Button in Maintain Centerv vb Concatenatelmages vb dvanced macros will be located in the macro directory Description Linearize laser attenuation AOTF or mechanical Automatic focusing according to a set configuration Bleaching of a rectangular area or a line combines old macros BleachRectangle lvbo BleachLine lvb and Spot lvb Color balance of Axiocam HRc Centers the field of view around the position marked with the cross tool Enables the combination of all kinds of images irregardless of the size and data depth to be presented in a time series Imported images can also be included Type of concatenation fitted to the smal
173. Ims re a e The slider near Pinhole Confocal Aperture SE enables you to change the aperture size of the es ee Power relevant channel tees SA ae Wo 532mm 30 dle The aperture size is indicated in um Optical P em oo aoe pl Slice and Airy Units The Airy value depends on the aperture of the objective excitations and the emission wavelength A small aperture size will increase the depth of focus but reduce the light intensity received by the PMT photomultiplier When you vary the aperture size an Optical Fig 4 56 Scan Control window Channels Slice value is displayed For optimum depth resolution Airy values should be small but in fluorescence applications not below 1 0 to keep the intensity loss within a reasonable limit A click on the 1 button sets the aperture size to 1 Airy unit A click on the Max button sets the aperture size to the maximum e The sliders and the relevant arrow buttons near Detector Gain Ampl Offset and Exposure Time enable you to set the photomultiplier of the selected channel during continuous scanning Detector Gain Setting of the sensitivity of the detector setting of image contrast and brightness values available between O and 50 Amplifier Offset Setting of the electronic offset background of the image can be set values available between 2 and 0 1 Exposure Time Setting of exposure time with display field for frame ra
174. Is activated 6 6 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan User Interface Carl Zeiss Gallery The Gallery is used as an overview of the images available in memory and their contents It is located just below the Tool bar Each small image represents a sequence The middle slice of each image sequence is shown The status bar of each image shows the name The name might be a number or a String Every image sequence has its own channel colour assignment see Display window When an image is copied the channel colour assignment is copied too Drag and drop techniques can be applied to copy images or define the function parameters Input and Output using the Gallery thumbnails e Position the cursor on an image in the Gallery e Press the left mouse button e Hold the mouse button down and move the mouse to the destination position e At the destination release the left mouse button the destination image will be overwritten To delete an image drag it move it to the wastebasket and drop it 6 2 3 Display Window This window is used to display an image sequence regardless of size or type To show multiple channel sequences each channel could have its own base colour The user can set these colours and the weighting tor each channel by pressing the corresponding button 2 at the bottom of the window To display a different image or image sequence it can be dragged trom the Gallery and dropped to the Display window
175. It is also possible to trigger an out signal via Trigger Control e Click on the required Set button e Open the Trigger out list box with a click on the arrow button e Select one of the trigger keys 1 to 4 e g Trigger4 In this example the relevant Set button is activated on pressing key 4 of the Trigger Control a marker is set in the Scan and an out signal given at the same time 03 06 B 45 0019 e 4 101 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 6 6 Time Series of a Frame e Set the relevant parameters for time control in the Start Series End Series and Cycle Delay panels e Start the Time Series with a click on the Start T or Start B button e If you use Trigger Control confirm the relevant Trigger key to start the Time Series with the first scan procedure e Use the Set 1 to Set 7 buttons to set markers during the scanning procedure which will allow you to evaluate interesting scanning images later cr Time end will finish time series even if you have created a program which would exceed the time end RS If a time series is interrupted before its programmed end the programmed number of images will be taken over in the database However only those images are stored which were created before interruption of the time series This is due to the fact that the original image parameters are to be taken over via the Reuse function If a stop time for time series is entered via the Trigger button or
176. LIVE Duo Scan Select Stand Database ile Es 5 2 Stand Select Database p NAIMSD atabase System_Contigurations_PASCAL mdb Browse Permanent Cancel OK Fig 5 3 Select Stand Database window If this is not possible proceed as follows The Stand Select tool permits a new or updated database to be assigned to the LSM 5 LIVE software program This function should preferably be performed by authorized service personnel e Close the LSM 5 LIVE software program and double click on the Stand Select icon on the desktop The Select Stand Database window appears on the screen The currently used database is displayed in the Database box Open EES Lookin E Database E e System Configurations Axioplan MDB System Contigurations_Offline mdb File name System Configurations Offline mdb Files of type mdb 24 Cancel Fig 5 4 Open window w Select Name Ed Name for icon on desktop OF ystem_Configurations_Otfline Cancel lcon Fig 5 5 Select Name window e Click on the Browse button to activate the new database The Open window appears on the screen e Select the directory where the new database is stored e Click on the name of the database file extension mdb and then on the Open button The Open window is closed and the name of the new database appears in the Database box e Click on the Permanent button The Select Name window appears
177. M 5 LIVE DuoScan 4 9 2 Settings Function The Settings function permits the individual setting and matching of software settings with regard to the following points Autosave Recording Reuse Database General Timeseries Database Table Viewer Scan Mean of ROIs Database Gallery Viewer Temporary Files Import Export Program Start Scan Information Hardware Image Status Display Image Display Toolbars Print Status Display Save 4 9 2 1 Open Close the Settings for user Window e Click on the Settings button in the Options subordinate toolbar of the Main menu This opens the Settings for user window e Click on the OK button to quit the window The last settings will be taken over Cancel enables you to cancel the procedure with any changes you made not being taken over Settings for user hpwdh i q Autosave Database General Database Table Viewer Database Galler Viewer Recording Reuse Timesenes Scan Mean at Als Temporary Files Frogram Stark Shutdown Import Export Scan Information Image Status Display Print Status Display Image Display Toolbars Save Cancel re Ed Startup configuration FITC Rhodarnir Don t show logo Hl Light manager Fig 4 201 Settings for user window 4 200 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Options Menu Carl Zeiss 4 9 2 2 Autosave ____Butasave Database General Database Table Viewer Database Galley Viewer C
178. Meaning of the Colours lt 420 nm gt Pink 421 475nm gt Blue 476 495 nm gt Turquoise 496 550 nm gt Green 551 590 nm gt Yellow gt 590 nm gt Red 03 06 B 45 0019 e 4 183 Carl Zeiss OPERATION LSM 5 LIVE Macro Menu LSM 5 LIVE DuoScan Important Information Optimize Fig 4 183 Optimize Macro If the calibration has been completed successfully with the Calibration Matrix DO NO LONGER USE THE PINHOLE AND COLLIMATOR FUNCTIONALITY OF THE LSM SOFTWARE IN THE MAINTAIN MENU The use of the pinhole sliders to raise the quality of the images is well known from the other LSM Systems On the LSM 5 LIVE only the Optimize sliders of the OptimizeLIVE lvo macro have to be used Fig 4 183 to compensate for misalignments e g temperature influences After the Calibration Matrix has optimized a particular objective the results will be stored automatically in a file as shown below These files can be found in C AIM BIN DO NOT CHANGE ANY OF TH VALUES 4 184 P 442340 pos Notepad File Edit Format View Help E iojxi SM 5 LIVE Calibration file Created or modified see File date and time 50 wl ef zoom phy collil colli2 es Ey 9 1 7 WWWNNNRRROSOSOoOSOSSSSeoooe 1 29s 9 39 9 29s 0 0 0 0 IS 0 0 0 0 0 0 0 0 0 0 Fig 4 184 Calibration file This file is valid for e ha ha lens Epiplan Neofluar 20x
179. N E EE 4 253 Aa E a ESEE E EE AA EEE E E E E E ETA 4 255 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Purpose Carl Zeiss A205 Coloca alon FUNCIO erouer e T 4 257 4 13 21 IS eo FO A EEEE E E ETETE A TOENE Anat E 4 261 AT FUNCT O eee 4 261 Aloa FPURCTOnN EEMETA N cana ante 4 263 Aale fe Fam DS 6 FAV E a E E E E A O E A 4 265 4 13 22 ISO D E E E O EEN 4 266 42A Dreco etnea a a ives esica Rumemer ees 4 266 l2 SEEMING Na TODAS esne EE EAEE A 4 267 4 13 23 Display s3 BAmagE VSA easa T A A 4 268 A2 Shadow ProjECUON ensinan a nE E a T E Eaa 4 269 AT 2322 Transparency Projectin secre E eoetaeagie ees 4 272 L2 SU ACOs OMG EINO soree r T E A dato Ma eimoserenndcetes 4 273 4 13 23 4 Appearance SCUUINOS 572 achcboranoctygssce teste ncvenadshagtd dase toaakscust ain beds iedtese Uabenuhbeactabenteiee 4 274 AWS 2 SONICS iid usu Athelt biel nsi dt once Yiaourainented brisk a Shaeriaiet nal ie see onnicd aot sible isda Donen Veanebvenieboldiatels 4 275 Aia 2A Display Topography Or LON ensine el este ae le lah beatae aoa ere nee and te 4 278 aE STATING SO 1S ClO E teeter ao tenn A E satel E T A A tse melanin oa nuer anaes 4 279 4242 Genere TODO aD caanacasecantuvicaiia a dasiialiolind death a aA 4 279 ANS 23 TOpodi PAYTA NOl e ae a iim vateoucune ads mee atutccgenadiatucs 4 280 ANS ZAG Processing OY FITEN creen r a A E innkeactanniseeemssamndenateds 4 281 AA DEPO MOJE aina E meena et 4 284 413240 GConext Menuo thesD Display ModE eer
180. Name column is deleted Opens the Track Configurations window A selected track defined in a Recording Configuration can also be stored as a single track for single tracking applications Also it s possible to load a single track in a multitracking configuration A click on this arrow button will move the selected track highlighted in blue one position upwards in the list box A click on this arrow button will move the selected track highlighted in blue one position downwards in the list box e Configure each desired track for Multi Track function as described for Single Track e For storing applying or deleting a Channel Mode Configuration use the Config button 03 06 Scanning an image A e Click on the Scan button in the Acquire subordinate toolbar to open the Scan Control window Cari The Scan Control window appears Fig 15 4 Scan Control x AEF te rae Yi es E an i BW Objective Lens Image Size amp Line Step Factor Objective Flan Meotluar 108 0 3 Frame Size 128 256 Al2 768 1024 Mode Channels e Select Mode in the Scan Control window Spot e Select the Frame Size as predefined number of pixels or enter your own values e g 300 x 600 in the Objective Lens Image Size amp Line Step Factor panel The number of pixels influences the image me n 512 resol ution Format m22 Scan Speed Ay FPS 4 TE gt Pixel Time 30 00 psec Scan Time 24 39 msec
181. No autosave This tab permits activation or deactivation of Rolie tea nine aati dior automatic data storage Only one option can be Export to MetaFluor format selected at a time Base directory C LSM510 IMAGES EJ Sub directory base name LSM Sub directory counter value 3 1 No Autosave On activation of this option the Autosave function is switched off Save and Save As give the same dialogues Fig 4 202 Autosave tab 2 Use LSM image database and auto increment image name On activation of this option newly recorded or modified images are stored by Save automatically and assigned to the name or defined in this function The image name is automatically created using a base name and a serial number For this a base name must be entered in the Base image name input box and a starting value for the serial number in the Counter value input box The Database selection box permits selection of the directory in which the data will be stored 3 Export to Attofluor format On activation of this option newly recorded or modified images are stored by Save in the Attofluor format The displayed Experimental directory selection box permits selection of the directory in which the data will be stored 4 Export to Metafluor format On activation of this option newly recorded or modified images are stored by Save in a subdirectory in the MetaFluor format An existing higher layer of folders must be selected for the subdirectory from t
182. O of O image entries see Image Database window page 4 19 The new image database can be used to store an acquired or processed image see Saving an Image page 4 29 L gt The start settings and the extent of the parameters displayed in the image database window can be changed as required via the Settings function in the Options subordinate toolbar see Settings Function page 4 200 4 18 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss 4 4 2 Open Existing Image Database Look irr a Database ce C The Open function allows one or several databases to be opened The images stored in the database s are displayed in thumbnail form they can be ge selected and loaded into the Image Display e window see page 4 224 Ea Network Neighborhood P J EJ My Briefcase e Click on the Open button in the File subordinate toolbar of the Main menu Filename Files of type Database Files mdb ERA This opens the Open Database window for selection of image databases Fig 4 9 Open Database window e f you want to load an image database from another folder drive directory click on the arrow button to the right of the Look in box This opens a drop down list box in which you can select from all available folders The window displays the various image databases with the file extension mdb e Open the image database by a double click on the respe
183. OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Acquisition of a Z Stack Once you have set up your image as defined in the above section you can collect a series of confocal images through the different focal planes of your specimen e Click on the Start button on the Scan Control window The system will automatically start the creation of a Z Stack Be careful not to bump the air table or the microscope until Z sectioning is completed Each successive Z Slice can be viewed by changing to the Gallery Mode This button is located on the right hand side of the image A black bar will be shown under the image and will move from left to right showing that the LSM 5 LIVE is in the process The laser will automatically stop scanning when the Z Stack is completed The entire stack of images can be saved using the Save or Save As buttons on the right hand side of the image r ai gri p Bn ti i i i i L 4 00 a en li ai j mie a a a 1 pmi f J i l a m T d k I 1 e HFI i LEI eri nE deri Fesi Slza Sta di Johane 0 be Fig 4 70 Image Display window of a Z Stack 4 86 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 4 3 Line T Scan Control HE In the Line mode fluorescent or reflected light along a freely definable line is displayed in the form of an intensity profile All the possibilities of creating an image Frame E Fone 1003 ef Z
184. PC 0 Oe EX Monitor 2 LSM 510 META e i Mi E3 Monitor aD AT _ ______ smsi 7 ta e ConfoCor 2 alls GigaStar Lvl m aas oe An Power suppl z Pply O BS a OO LVDS s gt ses J ee a m Safety e Cooling aggregate Z C E Fig 2 5 Multipoint connectors 03 06 B 45 0019 e 2 LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE Carl Zeiss Power Requirements LSM 5 LIVE DuoScan Q oCxwJo Z YY ait A QO 1 Key interlock Laser ON OFF 2 Door interlock interface Fig 2 6 Key interlock Laser ON OFF and interface for connection of door interlock The door interlock interface is covered with a green plug to bypass a door interlock To use the interface remove the top of the green plug and the bypass wire Then connect the wires of the door interlock at the same position Two door interlocks can be connected 2 8 B 45 0019 e 03 06 LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE Physical Dimensions Dimension of Shipment Crates Carl Zeiss 2 9 Physical Dimensions Fant em it em ei em ei eg Scanning Module LSM 5 LIVE Scanning Module LSM DuoScan Laser Module UV DuoScan only 140 o z z Pw Pugin unitextemal laser UV 6 3z z2 gt Power supply for Ar UV Cooling unit for Ar UV Water hose for Ar UV Fiber optic cable UV Fiber optic cable VIS ible Cables 2 10 Dimension of Shipment Crates L
185. Reduced Load with reduction in size Resolution Subset See a The Subset function allows images to be loaded Pixel ls P with reduced resolution For this purpose the Pixel y image pixels in XY Z are reduced It is also possible Plane 2 Cancel to reduce the number of slices in stacks and time Stack Time series e Click on the Subset button to open the Load Load 12 bit as 8 bit with reduction in size window e Enter one value each for n under Pixel x Fig 4 14 Load with reduction in size window Pixel y Pixel z and Stack Time in the Load every nth panel e lf required turn on the W Load 12 bit as 8 bit check box e Click on the Load button to load the selected images with reduction in size time slices and stack slices e Use Cancel to exit the window without any selection 4 4 3 6 Update Image Database Display Refresh e Click on the Refresh button to update the Image Database window 4 4 3 7 Copy Paste Image s to the CLipboard e Select the image s to be copied You can use the Shift and Ctrl keys for multiple selection e Click on the Copy button The image s is are copied to the clipboard and can be inserted in either the same or another database or in other software packages e Click on the Paste button to insert the image in the current database Identical to the WINDOWS function Drag amp Drop one or several images can be copied or moved from one database to another The F
186. SF The Point Spread Function describes how the light of a point object is distorted by the optical system This convolution makes the image appear grainy and structures in the image seem blurred This effect is most prominent in the axial Z direction as each lens is optimized for the two dimensional image of the object If the PSF is known it is possible to use mathematical algorithms to undo this distortion The image of the object is deconvolved using the PSF and the actual form is reconstructed Imaged Object Real Object Dekonvolution Fig 4 162 Point Spread Function PSF The effect of 3D deconvolution can be demonstrated impressively on objects with a known form As a rule fluorescent beads are used for this purpose The following figure shows the 3D deconvolution of an image stack with a fluorescent bead with a diameter of 1 um 03 06 B 45 0019 e 4 103 OPERATION LSM 5 LIVE Carl Zeiss 3D View Menu LSM 5 LIVE DuoScan Fig 4 163 Image of a fluorescent bead with a diameter of 1m before deconvolution A B and after deconvolution C D 3D Deconvolution I5 Click into window n w g fem Method Inverse Restoration Effect Weak SS Strong Auto detect Fig 4 164 3D Deconvolution window As the resolution of an optical system is Significantly lower in the axial direction than in the lateral X Y direction the greatest improvement in resolution can be achieved in the Z direction The
187. SM 5 DUO Inverted port config SP RP DuoScan Zoom 1 4142 RP SP LSM 510 Zoom 1 33978 Upright port contig Tube RP DuoScan Zoom 1 4142 RP Tube LSM 510 Zoom 1 25295 03 06 B 45 0019 e 4 187 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan e Choose Split image for the image window display You will see the scanned areas of both scanning systems and an overlay gt sea Read 512 512 2 channels 2 ba Fig 4 189 Split image e Choose LSM 510 or DuoScan to adjust the image match e While scanning move the sliders of the Image match macro to get a perfect overlay of the scanning and or manipulation system relative to each other e Offset X Y adjusts the horizontal and vertical image shift e Amplitude adjusts a size difference between the images e Rotation compensates for a tilt in one of the images 3 Store the final image and use the ReUse function for future image matching The Multitrack setup and the zoom factors will then be set automatically 4 188 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss 4 8 9 Visual Macro Editor The Visual Macro Editor is an optional software module which allows the user to program a variety of scanning procedures and image calculations as well as the combination of both 4 8 9 1 Open Close the Visual Macro Editor Window e Click on the Visual button in the Macro subordinate toolbar of the Main menu This
188. SM 5 LIVE DuoScan About 2 8 MB at 1388 x 1040 1 x 12 bit Win 2000 Win XP About 11 cm x 8 cm x 4 5 cm 2 3 x 3 2 x 1 7 3709 Blue anodized aluminum with cooling fins 1 4 photo thread for tripod mount CE cUL Via PC O 35 Celsius 10 80 relative air huminity no condensing tree air circulation required 7 8 2 High Resolution Microscopy Camera AxioCam HRm Rev 2 Cat No High Range Monochrome Number of Pixels Chip size Spectral range 000000 0445 553 incl digital interface and cable 1388 H x 1040 V 1 4 Mega pixel 8 9 mm x 6 7 mm equivalent to 2 3 With BK 7 protection glass up to 1000 nm with IR barrier filter BG40 limited to about 350 nm to 700 nm Selectable Resolution by Binning or Microscanning H x V 694 x 520 1388 x 1040 2776 x 2080 4164 x 3120 Dynamic Range Integration Time Cooling Optical Interface Size Registration Power Supply 7 14 Acquisition Time s 20 ms exposure 0 07 13 images s 0 2 5 images s Better than 2000 1 8 e readout noise 1 ms to several minutes Single stage Peltier cooling C Mount about 11 cm x 8 cm x 6 5 cm 2 3 x 3 2 x 2 6 GS CE cUL 12 V DC 1 A 230 V 110 V autodetecting B 45 0019 e 03 06 LSM 5 LIVE ANNEX LSM 5 LIVE DuoScan AxioCam High Resolution Digital Cameras Carl Zeiss 7 8 3 High Resolution Microscopy Camera AxioCam HRc Cat No 000000 041 2 312 incl digital interface and cable High Range
189. SM 5 LIVE DuoScan Fill Holes This function fills holes in all objects a Fill Holes fF EE Output 2 Hew Cancel Apply Fig 6 32 All holes in objects are filled by this operation Holes are structures which have a grey value of O and are Surrounded completely by voxels with a grey value not equal to O It is assumed that regions outside the image are black Holes which touch the image border are retained Parameters Input Input image sequence Output Output image sequence 6 42 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss 6 3 4 Functions in the View Menu Render Surface This function displays an image sequence according to the gradient shading method Surbace Alpha Input Output Mumber of Wiews Angle Angle Y Angle Z Channel Grey Low Grey High Aperture Reflection Auto Update W Show Cube Fig 6 33 ee EE 2 Mew m a e e as0 180 Start fo ef sea 180 End lo bl seo 180 OHAK ls O 255 es O e 255 e pf 1 10 jes a 1 100 E Cancel Apply The Surface tab sheet of the Render dialog window must be selected 03 06 B 45 0019 e 6 43 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan Method The Input sequence defines the data to be reconstructed If it is a multichannel sequence one or all channels can be selected for the reconstruction Output sets the na
190. SM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 10 Select Reuse This function allows to transfer acquisition parameters of an image trom the image data base to the Microscope control Configuration Control Scan Control and Time Series Control windows and applies those parameters directly on the system The acquisition parameters of an image are displayed in the Image Display window and can be viewed by using the Info function In the tab Image Status Display in the Settings function of the Options subordinate toolbar it can be determined what parameters to view with the Info function The parameters include the following Frame Size Speed Data Depth Scan Direction Average Zoom Rotation Offset Aperture Size confocal aperture Detector Gain Amplifier Offset Amplifier Gain Excitation Beam Path and Scan Mode Line Frame Stack Time Series However the required objective must be selected by the user e Click on the Reuse button The acquisition parameters of the active image stack are applied immediately to the system In the Options menu in the function Settings with the Recording Reuse tab it can be determined whether the objective should also be transterred and set Setting the microscope objective only works in microscopes with motorized objective revolvers 03 06 B 45 0019 e 4 241 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13
191. Saati te ain neste tae cane Acad One oar adams ace 4 186 48 8 Image Match Scantield Transformation MaCr0 cccecccceeccceeeeeeeseeeeeeeseeeeeeesenanneeees 4 187 4 8 9 Vedal Maaro TAO rT AEE AE a omnes aurbin de O nea EN 4 189 4 8 9 1 Open Close the Visual Macro Editor WINdOW ss sesisessinsssrnssiresrrrsrrrrerrrrerrrrerrreerrre gt 4 189 AiG 2 FUNCOM DESCHPUO uinen e E E R E S 4 190 AS Ed MaI O anana E A A T E EAO 4 195 4 9 Op UonsS MENUS canne ccsnecs 4 198 4 9 1 DYED FUNCUO N eresien EEA ER EEEE 4 199 4 9 2 SEAIS FUNC TO a E siete ceuteumierate en doeauteed ete cada deantaaeaeneeaeane 4 200 4 9 2 1 Open Close the Settings for user WINGOW cccccccccceeccccccee ee eeceeeeeseeeeeeeeeesaeeeeeeeeaas 4 200 A92 AWOSE eree en leah ctets foie tae tal etal gas dn Si rane a Clas Seah aatieie ale daddiidne T 4 201 LI Wats Gee alin te cote ee cere A rae ee let eh ccs Maen an set hace ahold 4 202 AO 7A Database Tabe Viewer EEE N a bare aa 4 203 A2 Daana e Gallen VONT riesta e A 4 203 A OA OT aa aa E A auueea cael aaeaes 4 203 718 S VO T a O aa E E A E E 4 204 4920 ImageState Dp a eeu a a Anon tn smecoe dasseaai dengan uabneeanacicasunhes 4 204 T2 PE a DE a a E E E O A E EE 4 204 4 9 2 10 ROC ONGING REUSE na E E E A E Sunktessaauy Oursataru 4 205 4 9 2 11 D OE a A A A E 4 205 4 9 2 12 Ny Can VOIR enr EE E taster crete OE A E E 4 205 4 9 2 13 TEMPO FES aee a a r octets ee sultanate ataben ed tt iaaedicdaatteet 4 206 4 9 2 14 P
192. Stack are also available in the Line mode Frame Size 128 256 Ale 1024 2048 The Line and Frame buttons are activated x alternately and exclude each other Bomal CAE e Set all the parameters for the Scan procedure Pe 1000 0 Frames per Second Mode and Channels or Z Settings in the same way as for the scanning of a frame or a Z Stack e Then click on the Line Sel button Number 7 Pixel Depth Scan Direction amp Scan Average Data Depth 5 Bit 12 Bit Mode Line Method Mean Zoom Rotation amp Offset Offset Offset s Offset Fig 4 71 Scan Control window Mode Line 03 06 B 45 0019 e 4 8 7 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 74 Scan Control zf 4 5 4 4 Camera Control Z Gelinas Close The use of this function permits the control of the Up a 7 external CCD camera settings Objective Lens Image Size amp Line Step Factor Objective Plan Meotluar 1070 3 Frame Size 128 256 512 1024 2046 ka 1300 Y 1030 Format 13001030 standard Channels 1 Open Close the Scan control window for camera control e Inthe Configuration Control window activate the Camera button Pixel Depth Scan Direction amp Scan Average Data Depth Bit 12 Bit Mode Frame l Method Mean Number 1 e Click on the Scan button in the Acquire subordinate toolbar of the main menu e Click on the Close button Zoom Rotation amp O
193. The simplest presentation of 3D information is a gallery showing the individual slice images sections of a stack arranged side by side with each slice apart from the next by a defined selectable interval on the Z axis Virtually infinite depth of focus The entire set of data can be imaged as a single projection The computer establishes an image composed of all in focus optical sections The image produced by this so called composite method has a virtually infinite depth of focus since the result is made up of information from in tocus planes only Rotary animation A sequence of projections is computed with the specimen being apparently rotated by a certain angle from image to image for example by a full turn about an axis If such a sequence is displayed on the monitor screen in rapid succession the visual effect is that of a rotating three dimensional object Stereo image pairs The computer establishes a pair of images corresponding to those we see with the right and the left eye respectively The two images forming the stereo pair can be shown on the monitor side by side They can be seen as a 3D image with suitable optical aids Another possibility is to present both images in registration with one image in the red channel and the other in the green one anaglyph Viewed through red and green color filters in a spectacle frame which only pass the image intended for the respective eye the two images form a 3D image in the brain Col
194. There is no depth cueing far objects would appear darker The illumination model is a Phong model 1 surface normal is determined for each Output pixel with diffuse reflection and specular reflection Diffuse reflection means that the surface reflects light with equal intensity in all directions The brightness of a given surface patch depends not on the view direction but only on the angle between light and surface normal Specular reflection is observed on shiny surfaces as a highlight The light is reflected as from a mirror The maximum intensity is observed when the view direction is the one of the mirrored light direction 03 06 B 45 0019 e 6 45 Carl Zeiss Render Alpha This function displays an image sequence according to the alpha rendering method Input Output Humber of Wiews li r lice Angle Angle r Angle Z Channel Threshold Ramp Max Opacity Auto Update If Show Cube Fig 6 34 3D FOR LSM LSM 5 LIVE Functions LSM 5 LIVE DuoScan la r 180 180 Start la r 190 180 End la t 180 180 fat 27s Bo ee ha af f fo 285 jis0 w f Ho 25 Cancel Apply The Alpha tab sheet of the Render dialog window must be selected One or more reconstructions of the input image sequence are computed according to the alpha rendering method This type of reconstruction should be used if there is no possibility to segment the structures in the image seq
195. Track Store Apply Single Track and Remove buttons permit individual tracks to be added saved or deleted Add Track StoreA4pply Single Track Fig 4 44 List of Tracks panel In addition this panel is used to activate deactivate the tracks for the scan procedure e To activate or deactivate one or several tracks for the scan procedure activate deactivate the check box of the relevant tracks The configuration of the selected track is displayed in the Beam Path and Channel Assignment panel e To select a track for the display of the beam path configuration click on its name The selected track is highlighted in gray or blue L When you switch from multitracking to single tracking the track selected in the multitracking mode highlighted in blue or gray is always transferred and automatically activated for the scan procedure All other tracks are deactivated and they remain deactivated when you switch back to the multitracking mode afterwards The following functions are available in the List of Tracks panel Add Track button An additional track is added to the configuration list The maximum of four tracks can be added One track each with basic configuration is added i e one ChL 1 channel is activated all laser lines are switched off emission filters and dichroic beam splitters are set in accordance with the configuration last used Remove button The single track previously marked in the List of Trac
196. VE Carl Zeiss Performance Features of the LSM 5 LIVE LSM 5 LIVE DuoScan 3 4 3 Computer Hardware and Software The LSM 5 LIVE is controlled through a standard high end Pentium PC Linking with the electronic control system is via an ultrafast interface The PC comes with the WINDOWS XP operating system The instrument is fully motorized permitting fast change over between methods as well as automatic operation Parameters once set or complex examination sequences once established can be saved and reproduced this way complete application programs can be loaded and executed by pushbutton control The software of the LSM 5 LIVE offers perfect facilities for individual settings of functions and parameters Conversion of the light signals into a digital image is effected by means of four 12 bit A D converters each of which can generate 4096 brightness levels The software provides a enormously wide range of image processing functions including all standard 2D 3D stereo projection functions same as sophisticated 3D reconstruction capabilities surface and aloha rendering digital processing of voxels and 3D measurement functions surface areas volumes As all files and images are recorded in MS Access databases elegant image database editing is just as easy as transferring the records to other programs 3 8 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan IMPORTANT NOTES FOR CHAPTER 4 Carl Zeiss IMPORTANT NOTES FOR CHAPTER 4 Blea
197. Zeiss File Menu LSM 5 LIVE DuoScan If required the filter features displayed in the work box can be stored e Click on the Add to List button The Add to List window will be opened Add Filter to List Filter Marne Pancal e Enter a name for the filter and click on OK The filter will be included in the Filter List panel ES Pm x Fig 4 16 Add Filter to List window Filter List panel Stack Convallaria All the stored filters are displayed in the Filter List panel and can be activated any time at a click of the mouse e Click on the name of the required filter The linked filter features will be displayed in the work box Fig 4 17 Filter List panel Filters which are no longer needed can be deleted e Click on the filter to be deleted in the Filter List panel e Click on Remove from List The filter will be removed 4 4 3 9 On Filter Function The On Filter function is a toggle switch to activate or deactivate selected filter settings 4 4 3 10 Delete Function e Select the images to be deleted from the image database e Click on the Delete button Confirm the safety inquiry then displayed by pressing OK The images and the acquisition parameters will be removed from the image database 4 28 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss 4 4 4 Save an Image to the Image Database The Save function allows to store an image together with the acquisition parameters
198. a 4 18 D EA Renee en er emer epee ered Teer teen ares 4 19 Saving ana Cr asccnasuianodiuadeteanaaen wees 4 29 Table TMOG G ni suicre Serarecras deaxahtiten ene Gd dnaucibeasuace 4 24 DECOM VOM ON trernen NR 4 163 Delete ge Ye 6 enee ee een pene een enone near eee eer 6 16 Object in specified size range 6666 6 41 DEDUNCOCING sb ccistacuseklohtamawheteatotatet 4 152 Detaching the Scanning Module 7 5 Detector GalN ceccceccceecee seen 4 319 4 320 DiglOGDOXCS araa 6 4 Dichroic beam splitters 4 56 Dilation 6 28 6 30 6 31 6 32 Display of WAGES caus ctunc ay toa een reaneaee 4 224 Display WINGOW masmorras 6 4 6 7 DY OS issen decide oeeee ual ynananieeiveosasisede 2 7 17 ANNEX LSM 5 LIVE Carl Zeiss LSM 5 LIVE DuoScan E Render Surtace iier esse ness 6 43 Save DiS PII AS oeron E 6 13 Edit TEM oeselnntencesuctactsntacerindadiedsesamerensctomeas 6 5 Save Image VaN S OPAD 6 12 EMISSION tE aa 4 55 7 2 G eeN 6 41 Erosion 6 27 6 28 6 29 6 31 6 32 Segment AutoMatlc n 6 35 TAS Segment lnteractive ccccccceeeceee scenes 6 33 EXC CO OMateaee acta E 4 59 os i i Set Channel Colour 6 10 Excitation laser lines 7 2 SMOOTH GAUSS na 6 24 Excitation of bleach track 4 113 Volume Features aaien 6 55 Pxiting tne LSM Progra nccueanasseellanntag 4 325 Full SCRO A aei GT 4 221 Exposure tIM cecce eee 4 75 4 89 4 317 G F Gale aere
199. a 4 251 6 3 6 7 E EST E E 4 15 4 17 Gauss filter renate asd iaa E E Sea 6 24 FHE menU eniinn TE O 6 5 Gradient shading method 6 43 Fills OES ices ais ce inhale tieed detebee ue whededenva venteiduias 6 42 Grey value segmentation Sey a Reto ee 6 33 6 35 FCS ERRENTA TE acide EEEN 4 57 4 130 FG EEEE ENE 4 318 4 322 2 EE E AE E SEE 4 68 4 71 FICO ANEA 4 223 creation OF a sereen 4 78 Pe DAIMO Mie aokerticn neen ines 6 6 FRAP GUIE ose teeeteteteteete te eeee tee neien 4 223 Pus COI re ie teat ereere 4 253 ee re Holes to fill sceeaarssanactanineedevetucecustes Geet dataset 6 42 ENING EE E AEE sia Arithmetics Add b spcnaciatsntint ntubveiaduaniiecaeas 6 17 Hyperfine Z sectioning sees ee Arithmetics Subtract 0 e ee 6 18 Automatic Object Measurement General6 58 BOOICAN AAG x ciucieetetacats tne te vadtondeies aie 6 37 Image BOO CaM MASK atastatit chariot tals tatiana see 6 40 Delete fUNCt ON oiccen 4 28 Boolean Not ccceececccecceeceeceeeeeeeeeeeaees 6 39 Display and analysis A N a ere 4 224 Boolean OFf ad iossiecnseied teens detec duns wacttestdees 6 37 EXO UOT anena 4 33 BOGIES OF poset incciuien pinnedindasettassciceions 6 38 MOO at gee ee ee eee ee ree 4 32 Condito ees 6 57 Information ccccccc ccc 4 311 Conas AUTOMATIC nishaieta crue anleee 6 21 oada N 4 19 COMPAS TINCT AGUNG ssania 6 19 Se 1G lec AE E aes ate eee ne 4 29 o A E mage acquisition 4 37 Delete All Images 0 n nonn
200. a a NEN COO ee E E E E E EE EE 4 227 PEE OO aa a S E E E E EEE 4 228 E N E A E E 4 229 DECC GIG E E A EE E A E ET 4 230 Functional Descrip CONG cesarean deve ce va mn serzue etme evtetassses E EAEE ear ESRDE 4 230 P ttons in the Ov rlay TOODA essiri EE tei sass 4 231 AEE O a E E E EE E E A ennernnceummiiactoiaacts 4 234 PMO E E E ates at ascanscanete eee ne aces 4 235 POVE E E A E AEE E A E E E A 4 235 SD e E E E EE T E E ee 4 236 IM E E E E E E E 4 230 Select Palette 0 cece cece ccc c cee e cece eee eeee teen cece riktnr rrtt Gene eeeeG dE EPEEEEEEEEEEEEEEEEEEEEEEEE EEEE EEn rEni 4 237 Editing and Storing a Palette secie aa aE EERE ANE E E aE A 4 238 Deea PACS e a E A E E aeivn tetaewants 4 239 EE B L i E E E E E A E E ee rere 4 239 LETAT E E EEEE E EE E E E AEA AREE 4 239 OCC E A E I EE E E E E A A E E E 4 241 Sa a o NE E A E E E N E N E EE 4 242 SOG ete R E E E E E 4 243 TE E E E E E e 4 243 ST NEOA a E A TE E E E 4 244 BESE NEE EE E E TET E E ETETE 4 244 Sey I E EE E NEE E E OEE A 4 245 Bisa ci I o A A E A geese ace E E eee 4 246 Ortho Select FUNC ON seinan aE NE OE i aE 4 247 Ormo Di tance FUNCIO sevesi n e E N E T NE 4 248 Ortho 2D DeConVolution FUNCTION cece cece cece cccce eee eeceee ee eeceeeeaeeeeeeueeeetesaeeeeesaeess 4 249 Pisae ean G10 een ene ee ee eee eee eee ee ee ee ee et 4 250 TSF Ny OT rats E A E tiated macs eters E E 4 251 Doy NS cece tas ene E E E A E 4 253 Display H O OVEN IEW seinere nE E TEE A
201. a the Shift function This image change can be stored in the image database via the Save or Save As buttons For applications requiring 3 or 4 channel scanning proceed in the same way as described for the 1 or 2 channel mode Convallaria single 7 Al Ready Alex S12 3 channels bit Fig 4 132 Image Display window with channel shift 4 6 11 Unmix The Unmix functionality permits to extract the emission of single fluorescence dyes e g GFP only YFP only etc from the overall emission band of strongly overlapping multifluorescence signal intensities by a pixelwise linear unmixing procedure Mathematically experimental fluorescence spectra of monolabelled samples are taken as an external reference Up to 8 different reference signals can be varied in this least square fit based algorithm to produce an 8 channel multifluorescence stack without any partial overlap between the channels 03 06 B 45 0019 e 4 137 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 11 1 Open Close the Unmix Window e Click on the Unmix button in the Process subordinate toolbar of the Main menu This opens the Linear Unmixing window e Click on the Close button to quit the Unmix window 4 6 11 2 Source Panel In the Source panel the image source for the linear unmixing process has to be defined This has to be a Stack a Stack Z series or a Stack T series Proceed as follows to select an image via the image selection box
202. ab permits determination of Temporary Fies the directory in which temporary files are stored C Use TEMP environment variable Use the following path 1 Use TEMP environment variable Temporary files are stored in the TEMP standard directory of the computer s hard disk Fig 4 213 Temporary Files tab 2 Use the following path The directory for temporary Tiles can be selected by clicking on the button in the Choose Directory window 4 206 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Options Menu Carl Zeiss 4 9 2 1 4 P rog ram St a rt Program Start Hardware Image Display Save Startup configuration FITC Rhodamin r The Program Start tab permits selection of a track configuration via the Startup configuration EE selection box which will be loaded automatically when the software program is started On activation of the Don t show logo b check box the initial screen with the Zeiss logo will not be displayed after the start of the LSM 5 software Fig 4 214 Program Start tab 4 9 2 15 Hardware Progam Stari Htodwere made Save D baim The Hardware tab allows you to set several Meela hardware defaults at the shutdown of the system eriou T miiran pib mal hih The Lasers off on Exit determine by activation of the Lasers off on Exit check box that the lasers are automatically switched off when the LSM 5 software is exited Allow lasers to cool
203. abels on the Axio Imager Z1 microscope with scanning module 03 06 B 45 0019 e 1 5 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss Warning and Information Labels LSM 5 LIVE DuoScan Warning LED is lighting up when laser is on OF IA R A 9 N amp 2 i A Fig 1 3 Warning and information labels on the Axioskop 2 FS MOT microscope with scanning module 1 6 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Warning and Information Labels Carl Zeiss Fig 1 4 Warning and information labels on LSM DuoScan systems LSM 5 LIVE DuoScan only 03 06 B 45 0019 e 1 7 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss Warning and Information Labels LSM 5 LIVE DuoScan Vor Offnen Netzstecker ziehen Caution disconnect gt power supply before openin D p g D brancher la fiche d alimentation secteur avant d ouvrir Carl Zeiss Jena GmbH 07740 Jena GERMANY Leistungsschild Rack CE Carl Zeiss CE Carl Zeiss Carl Zeiss Jena GmbH VISIBLE AND INVISIBLE LASER RADIATION S d 07740 Jena GERMANY AVOID DIRECT EXPOSURE TO BEAM Pi Ser Nr 2420xxxxxx Baujahr 20xx ECU LSM510 LIVE 000000 1298 270 2 N Ground 240 120V AC 50 60 Hz max 5000VA CLASS IIIB LASER PRODUCT i d C Laser 400 700nm 0 5W max output Carl Zeiss CE Ser Nr 2420xxxxxx ECU LSM510 LIVE 000000 1298 270 Carl Zeiss r Carl Zeiss Jena GmbH System electronic rack 07740 Jena GERMANY Ser Nr 2320Xx
204. able when inserted into the program flow using mathematical algorithms The new value is then used for any subsequent calculation in the program flow where the variable is part of the calculation e Click the Variable button on the toolbar to display the variable list Click once more to display the parameter list again e New variables can be added by typing in name and value in the next free line Toolbar buttons Run Starts the Macro starting with the firs block in the program flow column Stop Stops the Macro at any time Break Interrupts the Macro Clicking the button again will resume the actual program flow of the Macro Step Allows to go through the program flow of the Macro stepwise starting with the Tirst block in the program flow column Each click will perform the action of the actually highlighted block then jump to the next block which will then be highlighted Load Use to open a specific Macro and Save to store the Macro SaveAs allows to store the Same Macro under a different name Variables By clicking the button the list of defined variables is displayed New ones can be added by typing in name and value in the next Tree line Variables are assigned to Macros For each new Macro a new list of Variables has to be generated 03 06 B 45 0019 e 4 191 OPERATION LSM 5 LIVE Macro Menu LSM 5 LIVE DuoScan Acquisition Action Blocks i z Beam Path il i 4 Parameters e hil KA Scan il ail 4 a Beach Param
205. ace f Martan Maintain button Service mode for adjustment and setting of other parameters e g objectives 4 16 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss 4 4 File Menu The functions of the File menu permit images and the relevant information to be managed and handled completely in a database system You can also create your own databases The databases allow images to be stored loaded and deleted The additional functions Import and Export permit images trom other systems to be made available to the LSM 5 software or the export of images to other software packages The Print function allows individual or several images to be arranged on a print page for printout The Main menu can be ended via the Exit function In the pull down Main menu File two additional functions not displayed in the Main Menu Toolbar are available Clicking Recent Database opens up a choice of the last 5 databases opened by the program Clicking the appropriate line will open up that database A similar menu exists for Recent Image Files e In the Main menu toolbar click on File This opens another subordinate toolbar in the Main menu Mew Database Upen Database Save Save As Recent Databases 1 G Example Image Databases BioMed BioM ed mdb 2 G Example Image Databases M ateral aterial mdb Import Images 3 0 E sample Image DatabasesSNLOSNLO mdb Export Images Multi Print
206. acro from the Macros list box of the Recording panel e Click on the Run button to start performing the macro ILS Provided that a macro is linked to a button in the Macro subordinate toolbar you only need to click on this button to perform the macro Proceed as follows to delete a macro e Select the required macro from the Macros list box of the Recording panel e Click on the Delete button The macro will be removed from the list Proceed as follows to edit a macro e Select the required macro from the Macros list box of the Recording panel e Click on the Edit button The Microsoft Visual Basic editing window will be opened e Make the required changes also see the notes on page 4 172 4 8 2 3 Assign Macro to Button Function x f i Edit M Assign M B This function permits stored macros to be linked ee with one button each in the Macro subordinate Define Buttons toolbar cutter Re e Press the Assign Macro to Button button to Text Muti Time switch to the Define Buttons panel Project D AIM Macros MultTime MultiTime25 vb Bi Macras Newhacros A Macro Define Buttons panel Delete appv Proceed as follows to link a macro to a button of the Macro subordinate toolbar e Select the button number from the Button selection box e Enter the button labelling in the Text editing box e Select the name of the project file from the Project box using the button Fig 4 173 Macro Control window e Select the macro n
207. activated deactivated The Auto Stop function interrupts the calculation depending on the set image improvement delta between last but one and last cycle in no matter whether the value under Maximum Iterations has been achieved or not 03 06 B 45 0019 e 4 249 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Method POF 2 PSF tab optional with 3D DCV Numerical Aperture Objective 1 00 The objective data are displayed in the PSF tab In Magnification Objective 40 the case of wavelengths above 700 nm the NLO Refractive Index of Immersion Fluid 7 000 button Is automatically enabled The displayed values are always taken over by the Channel Ei system data but can be edited subsequently for NLO T simulation purposes Excitation E33 nm Emission E33 nm e Select the required method and determine the Pinhole 1 00 Aim Units relevant parameters The deconvolution calculation is performed immediately after the 2D Deconvolution window has been closed and the image display is updated on line Fig 4 257 PSF tab Cut 4 13 18 Display Cut Positioning the section planes ka a 2 Pitch aw This function allows to display a Z Stack of images at a user defined section plane cut plane improve the image of the section plane by trilinear interpolation The settings of Chan Zoom Slice Contr and Palette are applied e Clicking on the Cut button in the Display
208. ae tere eee een ees 6 56 ETOO ee p ends ale t eens bis 4 159 W Structures Dil E IA TA TEAT 6 30 WINQOW ssseeeesteeecseetessittesnnetesnnetciin 4 221 ENS arer 6 29 full SCrEEN vsssssssstsssntsrnnrrrrarrrrnrrrrnar een 4 221 Sse tages E E PAE AA ee 4 251 WINDOWS MENU eecseteseteeetciiee 6 6 Subtract image SEQUENCES ccceeeeee eee 6 18 X Switching on the enterprise UV laser 4 38 Switching on the System 4 12 KOM calculati Mieres iicstimaiacatonet 6 38 PE E L E E N S 4 244 7 20 B 45 0019 e 03 06 ANNEX LSM 5 LIVE LSM 5 LIVE DuoScan LSM 5 LIVE ANNEX LSM 5 LIVE DuoScan List of Key Words Carl Zeiss Z Zeiss TIFF image sequence 6 11 Z SECON xcactetatnar indi teddies naalnceaaueeietadt 4 81 ZS OUTING ia a E 4 79 EC K a ANN hee ea cee ieee en lied ae 4 79 CEE OVO tae oenar 4 86 LOOM eae aE E EE 4 228 03 06 B 45 0019 e 7 21 LSM 5 LIVE LSM 5 LIVE DuoScan Brief Operating Manual Release 4 0 March 2006 Contents Page Staring MNE SV SU IM AAAA AAIR 3 Settimo the MITOS CODE csi a E O AAEE A Eaa 6 Configuring the beam path and laserS nnannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn ennn 8 SPNO ANINA OE 11 SCOPING ANY NV AGC s s irs inae ni EnaA BEE iaar o a ETA ra iaai 15 Switching O the System ssscssak aaa dapada aiapidu paiia 15 Introduction For your safety Observe the following instructions The LSM 5 LIVE LSM 5 LIVE DuoScan laser scanning microscope inc
209. age Line frequency LSM incl VIS laser Max current Power Power Consumption Argon UV laser LSM DuoScan only Line Voltage Max current Power Consumption Class of protection Type of protection Overvoltage category Pollution degree 2 6 220 240 V AC 10 50 60 Hz 3 phases at 16A Phase 1 1 9 kVA max Phase 2 1 5 kVA max Phase 3 2 6 kVA max 5000 VA max 208 240 V AC 10 50 60 Hz 1 phase at 63 A Note For Line Voltage 220 V the con nector and power plug are rated for 63 Amps However wiring and fuse should be rated for 32 Amps 7000 VA max B 45 0019 e 100 125 V AC 10 50 60 Hz 2 phases at 25 A Phase 1 3 2 kVA max Phase 2 2 8 kVA max 5000 VA max 208 240 V AC 10 50 60 Hz 1 phase at 208 V 34 Amps 230 V 31 Amps 240 V 29 Amps 7000 VA max 03 06 LSM 5 LIVE LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE DuoScan Power Requirements Carl Zeiss oo 120 2 SEER S ES Se ore yes SiSas esx TE JO a tae Of Switchable multipoint connector Power supply 400 230 V A Phe YN Power supply f T 240 120 VA ie Power supply LM RGB OU scanner a ae Power supply too Power supply Ground wire User ports AWG10 green yellow mir 9 5 ERL Reserve ee iii el Sole FEI Reserve LSM DuoScan ere I 6 s Reserve LSM 5 LIVE T LM ext ue e Bo Stand Bo EZ
210. am Path and Scan Parameters It can be linked to the Data flow column If no connection to the data flow is made for example Display Image the result of the scan will not be displayed The parameters for bleaching the sample can be loaded from the list of bleach settings Bleach settings are assembled and stored in the Bleach control window of the main software This block performs the bleaching of the sample according to the bleach settings that have been defined and stored in the Bleach control of the main software Use the pull down menu to select the bleach setting from the list The list of properties lists all parameters that can be adjusted for the acquisition of the time series The value for each parameter can be typed in individually Clicking the Read Back button will take over all current settings from the main software The check boxes next to the parameters can be marked individually or clicking Use all or Use none they can be all checked or unchecked with one click Only the parameters with a mark in the check box will be taken into account and adjusted when the Time series block is set in the program flow column This allows to change only some of the parameters for example the used trigger at a certain position within the program flow as for each block the check boxes can be set This block can be connected to the data flow column B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss Windows Ac
211. ame IV Pixel Depth IV Stack Size Pixel IV Processed Size MV Date Time MV Scan Mode Fig 4 205 Database Gallery Viewer tab lmport Export Scan Information Image Status Display Print Status Display MV Use separate path for Import Save most recently used path at exit and reuse at next program start Use the following path at program start C SLsm51 Obilder conyallaria manual mdb IMAGES F User default path MV Use separate path for Export Save most recently used path at exit and reuse at next program start C Use the following path at program start CILSM510 Grafiken a User default path Fig 4 206 Import Export tab On activation of this option the path for the Import Images or Export Images and Data window can be entered directly in the relevant selection box or selected by clicking on the button in the Choose Directory window This path will then always be set when the Import Export function is used 03 06 B 45 0019 e 4 203 OPERATION Options Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Clicking on the User default path button firmly sets the C users default path prenenenensnanensesasenseeneeeseeeeneeensesenssnsssssssssssessens Import Export i Image Status Display Print Status Display Display the following information in the Scan Information window M Objective M Scan Time IV Free RAM IV Pixel Depth M Laser IV Free space on the pagefile M Stack
212. ame from the Macros box e Press the Apply button to assign the relevant macro to the specified button in the Macro toolbar 03 06 B 45 0019 e 4 171 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan Delete the linking of a button in the Macro subordinate toolbar Proceed as follows to delete the linking between a button in the Macro subordinate toolbar and a macro e Select the button number from the Button selection box e Press the Delete button to delete the linking 4 8 2 4 Editing and Debugging of Macros The Edit button activates IDE Integrated Development Environment which allows macros to be edited and debugged Under the Help Contents and Index menu item IDE contains detailed online help on its operation and on the VBA macro language Therefore only a few hints are provided in the following You should activate the required toolbars We would recommend you to activate the Debug toolbar via the View Toolbars Debug menu item The following buttons in the toolbar can help you when debugging macros b Starts running the command lines a Stops running the command lines 7 Interrupts processing of the command lines pause alll Sets a breakpoint in the line with the text cursor Mi Processes a command line and steps into subprocedures Processes a command line and steps over subprocedures t Exits the subprocedure step out gT Displays the value of the marked expression Watch
213. an 1 button Intensity values for ROIs and channels are shown in one diagram Chan button Intensity values are shown separately for each channel ROI button Intensity values are shown separately for each ROI Mono button Change between color and monochrome display of the intensity time diagrams Area button Display of the area of the ROI in the intensity time diagram depending on the set threshold values Area measurements of very small areas lt 10 pixels give only approximate values Mean button Display of the mean values of the relevant ROI in the intensity time diagram Chi Ch1 Ch2 Ch4 button Selection of the channel to be used Low 0 om Threshold low slider The intensity values below threshold are not displayed for the Area function High 255 m Threshold high slider The intensity values above threshold are not displayed for the Area function yack Buttons for Table functions Bee MERES iy Pa EGN EL Copy Table button The table of intensity values is copied to the clipboard Show Table button The table of intensity values is displayed on the bottom left of the Image Display window Save Table button The table of intensity values can be stored as a text file 4 314 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Image Optimization Carl Zeiss 4 14 Image Optimization 4 14 1 Single Channel Described b
214. an or equal to Threshold Mode Activating this option modifies the specification governing calculation of the Keep Maximum projection Threshold Pixel value at which the ramp rises variable from O to 100 Ramp Slope of the ramp variable from O to 100 0 corresponds to a vertical rise Maximum Opacity Degree of visibility at the top corner of the ramp variable from O to 100 0 corresponds to the bottom edge in the diagram Brightness The image can be brightened again by modifying the value from 0 2 to 5 Projection Transparency Mode C Masimum Transparent Keep Maximum Threshold Ramp Maximum Opacity 46 AB Brightness fi O i _ Fig 4 151 Transparency tab 4 7 2 4 Preview Panel IM Previews Slice The Preview function permits you to regard the eRe en E influence of parameter changes in an Image Display window Fig 4 152 Preview panel The Slice slider enables you to select the slice which shall be displayed in the Preview Image Display window 03 06 B 45 0019 e 4 157 OPERATION LSM 5 LIVE Carl Zeiss 3D View Menu LSM 5 LIVE DuoScan e After finding the optimum adjustment using the Preview function you have to generate the final version of the image using the Apply button The projection appears The computation can be followed in the image or by the progress bar ms The computed 3D sequence can be animated with the Anim button in the Selec
215. ancel e This stored ROI configuration appears in the Fig 4 77 Add ROI List window ROI Lists panel of the Edit ROI window 03 06 B 45 0019 e 4 93 Carl Zeiss TS Time Seres Control Start Series i Trigger Time FreScan Trigger out None Stop Series Manual Trigger Time Number 0 4 S gt Trigger odt None Cycle Delay Apply Store Delete Time Unit Trigger in None Trigger out None Marker Apply Store Delete Desciption Trigger in Trigger out Hone Hone Hone Hone None Hone Hone None Hone Hone None Hone None None Fig 4 78 Time Series Control window OPERATION LSM 5 LIVE Acquire Menu LSM 5 LIVE DuoScan 4 5 6 Time Series The Time Series Control window allows the definition of parameters for time series The Time Series function offers the following options for the creation of image series e Definition of break times between 0 1 ms and 10 hours e Determination of the number of steps from 1 to 10 000 for one scanning procedure e Setting of markers e Interruption of time control via pause function and resume of the time series function e Triggering of time series via numeric input external trigger pulses time of the PC 4 5 6 1 Open Close the Time Series Control Window e Click on the Time Series button in the Acq
216. and offset setting of images in the Scan control window before scanning Palettes are stored and retrieved together with the images when archived in the Image Database e Click on the Palette button in the Select toolbar The Color Palette window will be displayed e Select the required palette trom the Color Palette List panel by clicking on the relevant name e f you want to deactivate a palette selected before click on No Palette in the Color Palette List panel e Click on the Close button to close the Color Palette window e A changed image can be stored via the Save As function In the Options menu in the function Settings it is possible to switch to Mono automatically when a palette is activated and to Colour on deactivation of a palette In addition it is possible to activate deactivate Mono in the Channel toolbar Some of the handling functions of the Image Display window toolbars can be activated at the opening of a new Image Display window 03 06 B 45 0019 e 4 237 Carl Zeiss uted Fakie io Lei Asedh 512 9129 29 Jehareh 0 bi Display and Analysis of Images OPERATION LSM 5 LIVE LSM 5 LIVE DuoScan Lido ale be Color Palette List Ho Fgh Pherae acc F iie Scale Y Hardas Palette Fig 4 249 Image Display window Select Palette Color Palette and Add Palette to List window Color Palette No Falette Range Indicator Glow Scale Rainbow R ainbowz C Red C Green
217. and side of the Stage and Focus Control window Close button Start button Stop button 4 114 Ends the scanning procedure The Stage and Focus Control window is closed Starts the tile scanning procedure B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 9 2 Focus Position Panel Focus Position HR Focus a Work Te Load Focus buttons Z Moves 0 000 pm a Clicking on the A button moves the specimen stage nosepiece upwards Focus M Vi heel Clicking on the Z button sets the current Z position HRZ step um Focus step ym to zero joos 4 A far a gt Clicking on the W button moves the specimen stage nosepiece downwards Fig 4 102 Focus Position panel Focus Step slider 0 1 um is the smallest value which can be set and 100 um the highest e Clicking on the arrow keys changes the step size by 1 um e Pressing the CTRL key and clicking changes the step size by 0 05 um e Pressing the Shift key and clicking changes the step size by 10 um Work button Pressing the Work button moves the specimen stage nosepiece back to the Work position This is the position last set before the Load button was pressed Load button Clicking on the Load button lowers the specimen stage nosepiece to make it easier for you to change the specimen or objective Focus Wheel check box Clicking on this check box activates deactivates the focus wheel of the micr
218. anel to set the slowest acceptable exposure time speed The corresponding value for Frames per Second is shown below the slider e In the Number text box of the Pixel Depth Scan Direction amp Scan Average panel enter the number of measurements to be averaged i s Image optimization can be effected much faster if you select a smaller frame since less data have to be processed The greater the number of averages selected for Mean average Method the better the image quality will be the scanning time will be prolonged accordingly OPERATION Image Optimization Carl Zeiss Fide TOG x TOGA Z chari g b Fa pepe daa Dpi Zoom 1 Fae Ao Paeit Fig 4 314 Image Display window 74 Scan Control jel GA ee ig ROI Objective Lens Image Size amp Line Step Factor Objective Plan Meofluar 10s 0 3 Frame Size 128 256 Ale 1024 2046 s2 Yf 52 Format 512x512 Exposure Time SSS m 0 03 4 gt 10 3 Frames per Second Pixel Depth Scan Direction amp Scan Average Data Depth 5 Bit 12 Bit Mode Frame Method Mean scan Direction C pm Mumie E Offset Offset 0 00 um Offset r 0 00 um Fig 4 315 Scan Control window 03 06 B 45 0019 e 4 321 OPERATION LSM 5 LIVE Carl Zeiss Image Optimization LSM 5 LIVE DuoScan 4 14 2 Multiple channel 14 Configuration Control Channel Mode ambda Mo Online Fingerprinting 4 14 2 1 Requirements Sea sk Li e T
219. anning procedure and acquire an image Scanned images are shown in separate windows e Click on the Stop button to stop the current scan procedure If necessary Select Find for automatically pre adjustment of detector sensitivity Select Fast for continuous fast scanning useful for finding and changing the focus Select Single for recording a single image Select Cont for continuous scanning with the selected scan speed Select Stop for stopping the current scan procedure 03 06 Image optimization Choosing a lookup table e Select Palette in the Image window of the scanned image Fig 18 The Color Palette window appears e In the Color Palette List panel click on the Range Indicator item Fig 19 The scanned image appears in a false color presentation Fig 18 If the image is too bright it appears red on the screen Red saturation maximum If the image is not bright enough it appears blue on the screen Blue zero minimum Adjusting the laser intensity e Set the Pinhole to 1 Airy Unit Fig 20 e Set the Detector Gain slider to a middle position e When the image is saturated reduce AOTF transmission in the Excitation panel Fig 20 using the slider to reduce the intensity of the laser light at the specimen Adjusting gain and offset e Increase the Amplifier Offset until all blue pixels disappear and then make it slightly positive Fig 20 e Reduce or increase the De
220. appear e Meet the requirements listed in the Requirements for Adjustment window and press the OK button Aperture adjustment will then run automatically The adjusting procedure takes approx 3 min The determined data are stored automatically and will be available for all further examinations using the same contiguration OPERATION Maintain Menu TS Question Fress 0K when ready for adjustment do not disturb the adjustment actions Fig 4 231 In Configuration Control On the microscope Carl Zeiss Activate ane track only Select the excitation line 486 recommended Activate the proper laser Select the matching channels One detection channel onlyl 10s objective i recommended Switch polarization slider to None position Use thin fluorescence specimen Focus to specimen Requirements for Adjustment window e The Move To Stored Position button enables the collimator to be set back to the factory adjustment e Activate the Fast Adjust Mode check box for a faster readjustment Ie 03 06 A change of the aperture size made manually in the Pinhole panel will not be activated in the Scan Control window Therefore changes must always be made in the Channel Settings panel of the Scan Control window A filter change in Autoadjust is not displayed in the Config Control window Please do not make any program manipulations while the automatic aperture adjustment is running status
221. ar below when this option is activated Threshold All brightness values below the Threshold range O to 255 are ignored or treated like O when determining the depth and the display Contrast Defines the factor with which the contrast of the overlaid series affects the contrast of the depth coded color Brightness Defines the factor with which the brightness of the overlaid series affects the brightness of the depth coded color Display Scale Bar Displays a colored scale in the image Display Grey level The depth information is displayed in gray levels 03 06 B 45 0019 e 4 153 OPERATION LSM 5 LIVE Carl Zeiss 3D View Menu LSM 5 LIVE DuoScan 2 Transparency tab Mode Maximum The color is defined by the z position of the brightness value Mode Transparent The transparent projection is built up from the rear to the front The color is defined by the Z position at which the original was last higher than or equal to Threshold Mode Activating this option modifies the specification governing calculation of the Keep Maximum projection Threshold Pixel value at which the ramp rises variable from O to 100 Ramp Slope of the ramp variable trom O to 100 0 corresponds to a vertical rise Maximum Opacity Degree of visibility at the top corner of the ramp variable from O to 100 O corresponds to the bottom edge in the diagram Depth Coding Transparency Mode Maximum Transparent Keep Maximum Thresh
222. ards 4 4 7 2 Printing Images To print several images on one page proceed as follows e Use the Overlay functions to additionally illustrate the graphics and images to be printed e Select the paper orientation by clicking on the Landsc or Portrait button e Open the image to be printed or select it from the relevant image database e Click on the Copy button The image is copied to the clipboard e Inthe Print AIM window click on the Paste button The copied image appears in the work area of the Print AIM window You can click on it with the mouse and move it to any position on the print page or you can adapt the image size e Proceed in the same way with all other images you want to print e Finally arrange all images on the print page as required e Click on the Print button to start the printout e Close the Print AIM window by clicking on the amp button 4 4 8 Exit e Make sure to save all required images in the image database or export them e Close all open windows of the LSM program by clicking on the closing icon in the top right corner of each window e Click on the Exit button in the File subordinate toolbar of the Main menu The LSM 5 LIVE Main menu will be closed and the LSM 5 LIVE Switchboard menu appears on the screen 4 36 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 Acquire Menu e In the Main menu toolbar click on Acquire This opens a
223. are Rasterizer ATI RADEON Mobility DELL Software Wertes Processing Version 8 DirectSD Hardware ATI RADEON Mobility DELL Software Vertes Processing Version 5 Render board features to use Iv Palette textures for single channel images IY Intensitydlpha textures for single channel images M y textures only IY Enable display lists Use up to MBytes for Textures Default Fig 4 294 3D Renderer window 03 06 B 45 0019 e 4 293 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 24 7 Measurement Functions The topography measurement functions are activated via the Measure button bar The measurement functions can be performed in the 2D or 3D display mode Automated convention in height statistics analysis lt 9x9 2D protile Pxx RXX WXX The following measurement functions are available Diagram button Diagram display The Profile z Histo Curve of tp and Grad Histo diagram display modes can be activated via the Diagram button and deactivated via the Off Diagram button By activation of the Diagram button an additional button bar is displayed for the selection of the required diagram or for deactivation The labeling of the Diagram button changes depending on which diagram display mode has been activated Ot Profile 2Histo Curve of tp Grad Histol Roughness button Calculation of the roughness parameters Roughn gt Volume bu
224. astebasket all of them can be deleted by a single function If any image or image sequence is needed for further use save them first Parameters None 6 16 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss 6 3 3 Functions in the Process Menu Arithmetics Add This function adds two image sequences Add Subtract Input 1 jao AT Input 2 ao A Output Booo Hew e Wrap Clip Normalize Cancel Apply Fig 6 11 The Add tab sheet of the Arithmetics dialog window must be selected If one or both input sequences are multichannel sequence any number or combination can be selected The number of selected channels for Input 1 and Input 2 must be the same They will be combined from left to right This function adds the two image sequences Input 1 and Input 2 voxel by voxel and generates the image sequence Output Note that a resulting grey value may be greater than 255 4095 The parameter Mode determines how a range overflow is handled 1 Wrap No normalization the grey values are displayed modulo 256 4096 If the result is greater than 255 4095 the value 256 4096 is subtracted from it 2 Clip Grey values which exceed 255 4095 are replaced with 255 4095 3 Normalize The resulting grey value range is scaled to the range 0 255 0 4095 Parameters Input 1 First input image sequence Input 2 Second input image sequence Output Output image sequence Mode 1
225. aw of an ellipse in the Image Display window first click sets the center point displayed line permits determination of the extension second click sets the first dimension then the second dimension and the rotation direction can be determined third click sets the second dimension and direction and ends the procedure Circle button Draw of a circle in the Image Display window click and keep the mouse button pressed to set the center point drag the diameter let go off mouse button again to end the procedure Polyline button Draw of a polyline figure in the Image Display window first click sets the starting point each further click adds a line double click on the starting point closes the figure and ends the procedure Yo e a Oe Recycle bin button All the ROIs dragged to the scanning image are deleted If an area outline was marked before this area is now deleted in the scanning image e F Auto Color button A defined color from the list of colors can be assigned to the ROIs In that case the same color is assigned to all the individual figures In the Auto position the outlines of the dragged ROIs are automatically colored differently Line button This button allows you to determine the line thickness of the area outline This is for display purposes only The scanned line is not effected Fit Frame Size to bounding Rectangle of all ROIs check box If this check box is ticked the scan procedure is displaye
226. axis right handed coordinate system Alice is an individual image in a sequence of images The numbering of the slices starts with 1 Image sequences can consist of several channels Most functions and the Display window are providing buttons to select all or a subset of channels stored in the selected image sequence The Output image sequence will only get those channels which are selected on the input side The button selects all channels in the image sequence to be used clicking with the left mouse button on it Clicking with the left mouse button on any of the number buttons toggles the state of this single channel Clicking with the right mouse button on any of the number buttons selects this single channel exclusively All other channels are deselected 6 2 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan User Interface Carl Zeiss 6 1 2 The Image Properties Every image sequence has its own set of properties They contain the scaling and the scaling units The scaling and its units are required for 3D reconstruction and measurement If a sequence of LSM TIFF images is read in the image properties are loaded automatically from the file header and allocated to the image properties of the new image sequence 6 1 3 Memory Usage All images shown in the Gallery are currently loaded in the system memory of the operating system Some functions need additional temporarily used memory during their execution If the me
227. ay Topography for LSM This optional function allows to process display and measure topographic information use frame Z Stacks and frame Z Stacks over time The Topo function is mainly used for applications in material research and industry The settings of Chan and Zoom are applied The settings of Slice and Contr are not applied The Palette settings are applied in some 3D display modes Click on Topo will display the Topography toolbar All changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed e Click on the Topo button The Topography toolbar is displayed The topography of a Z Stack is displayed in the Image Display box of the Image Display window The parameter used at the last exit of the Topo function are applied Pasg 5124512015 2chennah Oa Fig 4 280 Image Display window Topography display 4 278 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss The Topography toolbar contains the following function elements Channels buttons The selection of a channel to be used Generate buttons The selection of the mode of calculation of the topography image maximum center of gravity First Last Threshold buttons The selection of thresholds intensity height Auto Z to be used Filter buttons The definition of filter procedures geometrical frequency cut o
228. buttons in the left group start and stop playback of an image sequence al Reverse playback Forward playback Play forward and then backward again jojo m Stop playback al Pause playback The buttons in the middle group control the settings of the current sequential image t Reset to start slice a Single step backward 1 sequential image each regardless of Increment pe Single step forward 1 sequential image each regardless of Increment Set to end slice Increment Image increment Wait Time Displays delay between two images in milliseconds 03 06 B 45 0019 e 6 9 3D FOR LSM Carl Zeiss User Interface Set Channel Colour This function sets the colour and weight for the channels Set Channel Colour Image E Weight 0 2004 Channel 1 ho ore Channel 2 ho ee Channel 3 ho We Channel 4 fo ef S si 0 Channel 5 ho yf 0 Channel 6 ho Ff _ 0 Channel 7 ho Ff ss 0 Channel 5 ho Ff Ss Dies Cancel Fig 6 5 200 200 200 200 200 200 200 200 I D JEN x OOCOUTEON LSM 5 LIVE LSM 5 LIVE DuoScan Each image sequence can get its own colour definitions All functions will inherit the colour definition from the Input sequence to the Output sequence By default the colours are set to 100 weighting and the pure base colours red green blue are defined The weight can be any value between 0 and 200
229. can Macro Menu Carl Zeiss Project Lsmi AO503 New Load cave Savecs Macros Edit Delete E ditor Fig 4 170 Macro panel Macros are stored and managed in project files lvb Before you can record or edit a macro you have to create a project as follows e Press the New button to create a project file Anew project is created and displayed in the Project selection box e g LSM 150503 The project name is automatically default but can be edited afterwards To activate an existing project proceed as follows Open HE Look ir a Macros f poe FEE e Press the Load button The Open window will be opened e Select the relevant project file data extension Ivb from the Macros list box Click on the Open button The project file will be opened and the oo 6 macros contained in it are displayed in the oo Tr Macros selection box of the Macro Control i Open a read only window Recorded macros are stored in main memory first Fig 4 171 Open window Before the macros can be assigned to the buttons in the Macro submenu the project must be stored on the hard disk e Press the Save button under the project name in the Macro Control window and determine the file name in the Project selection box if required 03 06 B 45 0019 e 4 169 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan 2 Recording panel Before recording a command sequence you can enter the nam
230. ccccccccecceeeeeeeeeceeeeeeeseesneeeeeneeees LOM S LVE me B10 6 S cr ga Pane leirasara iraan ee enn ee SCAN Control o oo cece ccc ccc cece ccccccecucucucecececeueueucucucucsecececececeueueueueeeass Open Close the Scan Control WINdOW ccceceeeecceeeeeeeeeeeeees PVC cae sess see ek cuca theese yates oe eee denon iecch nce cet teecninetnaee cuentas Edit ROI Region Of INterest cece cecececceeeeeeeeeeeeeeeeeeeeneeeeeaaens Open Close the Edit RO WINdOW cccccceccceeeeceeeeeeeeeeeees FUNC HOM DSCC TOL scaenaesarussasteniaeatere accameut anoa NE E ENE Time SON OS ersertarerecre essere tes nanai ye aceeteae araara ekee EnA KEKERE E E eaavceeeaees erate Open Close the Time Series Control WINdOW s src Start Series es eee ne eee ee DEO Cie ee IC ici psc ersz ecrcreccosc T E A Cycle Delay Time Interval Panels ccccceccccceeeececeeseeeeeeeene ees Marker PS CMa sree tens ccce bee tose dvestea tiedesscarsddaondeteceamdua ined suamndetssaae tasted Time Series Of a Frame oo cece eecceeccecceeecectececeeeeeeeeceeveeeereeeeeerernenes Time Series of a Frame over Z Stack option Time Series with Mean ROL ccccceceecceceeeeeeeeeeeseneeeeeneeereaeeneeaes Stripe bleach function LSM 5 LIVE without LSM DuoScan Edit Bleach LSM 5 LIVE DUOSCAN 1 0c ccccccccccceccucecceccuceececeeeuceeee Open Close the Edit Bleach WINGOW 0cccccecceeeeeeeeeeeeeeeeeees SENS FG E E E E E EE E
231. ce 1 Wrap 2 Clip 3 Normalize 4 Shift Clip B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Contrast Interactive This function allows interactive changes of the contrast of an image sequence 4 Contrast E4 Interactive Automatic Linearize Input Moo ma Dutput 20 o Nfe Channel A 1 Clip Grey Values M Input Histogram Lje al fies ale H ps a a5 utput Histogram iz ee L la h E 127 kit H 255 KG 0 255 Fig 6 13 The Interactive tab sheet of the Contrast dialog window must be selected A grey value range of the Input image sequence is scaled to another range in the Output image sequence Both ranges can be edited interactively This function is used to achieve a better view of an image sequence or to scale a range of grey values to single value for a special coding in an image sequence The function does not improve the result of the linear segmentation function Segment Input indicates the sequence to enhance If it is a multichannel sequence a single channel all channels or any number can be selected The Input histogram shows the grey value distribution of the selected channels of the Input image sequence Output defines the name of the result sequence It will get only those channels which are chosen by the Input parameter The buttons labeled with 8 and 12 define the grey value intensity resolution in bit Normally the result will get th
232. ce 60 2 switching positions for switching to 100 mirror or to interface for P amp C modules Adapter Video 44 C 2 3 0 63x Cat No 452997 0000 000 This adapter is needed for attachment of the high resolution AxioCam microscope cameras on the Axiovert 200 M SP gt No other cameras are supported by the LSM Software 7 16 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 7 9 List of Key Words Numbers DAD ses ta ica tant dictates cots see saa 4 266 ID EE AEE Gaeta E E cit asst aseae entices 4 268 3D for LSM Terms and deftinitions ccccccceeeeeeeeees 6 2 BDE o A E A at E 4 15 4 152 A ADOUTESM S UVE re 4 223 PCT OG ALC saiia ra a 4 55 PACOUM Oo ccdsnpattctientataiaattaeteda nckadschoaes 4 15 4 37 Add Image Sequences 6 17 Alpha rendering method ccccccceee 6 46 PING CAN AO Merce acc nercete N 6 37 ARRO oasi cer decent tena see nines duis are gaat he 2 PIM CCA ase a TO 4 319 7N 9 7 8 PRC eina ee 4 321 Analysis Of iMages ccccceecceeeeeeeeeees 4 224 ANIMAU erant ere aana iA tector 4 239 AO a A 4 254 Automatic object measurement 6 52 AVEA Genen 4 73 Axio Imager control arvaracncnuis eatidnimavurdaiencatt 4 41 Axioskop 2 FS COU OW werneri 4 50 AXIOVETE CONU Ol earnan ctetucem tere 4 46 B peant Dat i 4 55 4 57 4 59 EAEE EAE E A E 4 60 4 316 4 322 IE e E AET PE E EE tact E SE 4 110 Bleach parameters a a 4 112 DIC ACM OGIO S ireua 4 112 C Calculation
233. cess Menu Carl Zeiss 4 6 Process Menu e Inthe Main menu toolbar click on Process This opens another subordinate toolbar in the Main menu ALSH 5 LIVE El Ei File Acquire Process 3D View Macro Optons Maintain Window EditUl Help File rire lon Cone Multiply Fig 4 106 Process menu The functions of the Process menu permit already stored scan images to be subsequently linked and processed using mathematical functions and algorithms 4 6 1 Add i Add EJ Close Click into window The Add function links two channels each of one or two images into a new channel through addition The channel created in this way can be Apply stored via the Save As function Convallaria Tz 4 6 1 1 Open Close the Add Window e Click on the Add button in the Process subordinate toolbar of the Main menu Destination This opens the Add window e Click on the Close button to quit the Add window New Image 8 bit Channel New v Source 1 14 00 Source2 4 00 y 100 00 8 00 VV Preview Fig 4 107 Add window 03 06 B 45 0019 e 4 121 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 1 2 Source Panel Click into window In the Source 1 panel the first image source for the addition process is determined The current ae Channel ont F image is displayed in the display box of the image ee selection box Fig 4 108 Source 1 panel Proceed as
234. ch series Fast Z stack and line scan acquisi tion Piezo objective focus units 03 06 When doing bleach series on LSM DuoScan systems the time stamps and bleach markers indicated in the images will depend on the start position of the scanners in the image frame Since these are not identical for the imaging and the photomanipulation scanners sometimes imaging seems to start too early after a bleach event Ignore the last two digits in the time stamps and bleach markers in this case When using the modify images macro to convert time series data into Z stacks an arbitrary section step of 1 um is applied to the stack This will also lead to an arbitrary time stamp when reconverting the Z stack into a time series The order of the images however is preserved under all conditions When acquiring fast Z stacks with line scan mode the starting point of the stacks and the vertical dimensions can differ depending on uni or bidirectional scanning mode This is a mechanical effect which increases with rising line scanning speed Avoid extremely fast speeds in the hundred s of frames per second range when acquiring Z stacks The piezo objective focus units 1325 210 1325 212 and 1325 213 with an increased travel range of 250 um are controlled by a 14 bit logic Due to the increased travel range this implies a smallest step of 15 nm in contrast to former fine focus solutions with a smaller travel range but a smallest step size of lt 10 nm
235. chameleon NLO laser in case the laser is falling into CW lasing and does not get to mode lock anymore Reset_live_servos vb Reinitializes all servo motors of a LSM 5 LIVE to overcome possible warmup deviations trom default positions Scalebar lvb Indication of self defined intensity levels assigned to a ROI as scale bar in the image also attaches tick marks and concentration values to the grayscale color wedge ScanfieldTransform vb Adjusts LSM 510 or LSM 5 LIVE image match relative to each other or to LSM DuoScan ROI s SphericObjectiveCorrection vb Calibrates the spheric component of objective error by automatic acquisition of a Z stack on a flat surface and a spherical fit of the topographic data Projects a testgrid into the image window password protected 3 During installation default macros will be installed according to their type either in AIM AIM HWT or AIM Macros Self generated Macros will be in AIM Macros 4 174 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss In case of a new installation old macros will be stored in AIM Macros BackupMacros or AIM Backup to avoid problems with identical names of existing and new macros 4 8 4 Sample Macros The LSM 5 software package includes e g the Distance Profile Multiple Time Series and StitchArt plus sample macros They can be easily executed by clicking on the relevant button in the Macros subordinate toolbar During the executi
236. ciaccsectenemsectctteaetdacnatnts eee ned rhc eteiapttaeetuescnaapedaigeoasacaprmeendestdgnssntanasel 4 165 IVT ACI O MENU agate toates rtiopesnespaanccsuasatensnors were vedatan ane N 4 167 KELo RE ielo a E E seomceras E T AE A N sar T T 4 167 MTO CO O en E A E E A AE E E E 4 168 Open Close the Macro Control WINGOW ccccccceccce se ccceceeceeeeeeeeseeeseeeneeeeeeeeeeeeneeenas 4 168 FGI MaG O OCT ee carvers E E E EE 4 168 Assign Macro to Button FUNCtION 5 ceusecyrscerencossqurs eacnsastuanectiotteontan cosecrrtecesiosscaameaytesentanss 4 171 Editing and Debugging of MAaCrosS eccdevacedecarstenediianherteedsien dxedniacusiveetiussGtiealesseealdmdeceonaes 4 172 Overview of Available Macros a aecereeai cesses aterrsct ctertsecisapmnsctateedateenia la anata rrr re rrrE e rrr Ee rnrn ne rerne 4 173 Sea e e EEEE EE EE EEEE eee ee ee eee 4 175 DO N O E a aan E E A ER E O E 4 175 Profile MACO ssnnnessnnnessirnuesriresrir e rintt rir t rrr t nnr ssannestibeneseniadannseegeseeepancuet sb etentee ke 4 175 Multiple Time Series WIA CY Ose cttsccarrstta ce senraacint gather aste err needatathpioinaatnnaedecedindsdepinsnadindauatnes clematis 4 176 IOI te eects oe A E E E E E ees 4 177 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Purpose Carl Zeiss 4 8 6 ODUNSI Oanei e e gaa lertiaud send atteau ale ad eat pieend edo tainted etic cedaicton 4 186 4 8 REIN IC IO ren na tenls directa a ed tack Rion ch aavcen c a du Pate dee aunt Daan
237. ck on the On button Status ready On appears 03 06 B 45 0019 e 4 39 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan e Use the Output slider to set the laser power which is ideal for the measurement job Thus the laser needed for image acquisition is available Argon Set output between 25 and 100 of the maximum tube current Optimum operation is at 8 A lowest laser noise However the laser life is reduced if the laser is constantly operated at 8 A Therefore 8 A should be used only if this is absolutely necessary Enterprise Set output between 50 and 100 of the maximum tube current Optimum operation is at 20 A Tube Current lowest noise However the laser life is reduced if the laser is constantly operated at 20 A Therefore 20 A should be used only if this is absolutely necessary To switch on the Enterprise laser proceed as follows e The internal water cooling LP 5 is running e Start the PC wait until NT system is booted e Switch on the power supply of the Enterprise laser power potentiometer turned to maximum e Start the LSM 5 software 3 Please bear in mind that a cooling phase of at least 5 minutes is required between switching off of the laser via the software and switching off of the entire system via the REMOTE CONTROL main switch or the Power Supply switch of the Enterprise UV laser If the LSM 5 software is already running and you want to use the UV laser do the following
238. cl AxioVision AC digital interface cable and IR barrier filter BG40 enclosed 1388 H x 1040 V 1 4 Mega pixel 6 45 um x 6 45 um 8 9 mm x 6 7 mm equivalent to 2 3 Number of Pixels Pixel size Chip size Spectral range With protection glass limited appr 350 nm to 1000 nm With IR barrier filter BG40 appr 350 nm to 700 nm Increase of IR sensitivity Approx 17 000 e NIR Mode Max Full Well Capacity Selectable Resolution H x V 276 x 208 Binning 5x5 346 x 260 Binning 4x4 462 x 346 Binning 3x3 694 x 520 Binning 2x2 1388 x 1040 single shot Live frame rates depending on hardware and software configuration H x V 1388 x 1040 1 694 x 520 2 462 x 346 3 Readout of Sensor Sub Regions ROI Digitalization Dynamic Range Integration time Cooling Control signals Interface Optical Interface Thread depth for objectives 03 06 Binning Factor Frame Rate 20ms 7 13 17 Adjustable 12 bits 18 Mhz pixel clock Typical 1700 1 ati 10 e readout noise 1 ms to 205 One stage Peltier cooling TTL output for controlling of external shutters PCI interface card 32 bit 5 V with one cable 5m for data control and power supply C Mount max 5mm B 45 0019 e 7 13 Carl Zeiss Max Tile size per image Operating Systems Size Weight Housing Registration Power Supply Environment conditions ANNEX LSM 5 LIVE AxioCam High Resolution Digital Cameras L
239. computed and the result indicated under Area Measure e f you specify a top and bottom intensity threshold the area lying within this intensity interval can be computed 4 256 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss e Specify the thresholds either with the Threshold low and Threshold high sliders or with the aw buttons 4 13 20 3 Colocalisation Function The Colocalisation function permits interactive analysis of two channels of an image by computing a scatter diagram colocalisation e Click on the Colocalisation button The scatter diagram is created and displayed beside the image e Click on the xy button of the Display toolbar if you want to return to the original image How a scatter diagram is generated All pixels having the same positions in both images are considered a pair Of every pair of pixels P1 P2 from the two source images the intensity level of pixel P1 is interpreted as X coordinate and that of pixel P2 as Y coordinate of the scatter diagram The value of the pixel thus addressed is increased by one every time up to the maximum number of pixels used This way each pixel of the scatter diagram is a value that shows how often a particular pair of pixels has occurred 3 Differences between the images cause irregular spots in the scatter diagram Identical images produce a clean diagonal line running from bottom left to top right because only pixel
240. ctive key icon e g Convallaria mdb or click on the name of the image database for selection and open it by clicking on the Open button This opens the image database window e g Convallaria mdb AIM in which you can select from a variety of options e Click on the button in the title bar of the Database window to close this window and make no selection 4 4 3 Image Database Window The Image Database window allows you to choose one of three different display modes Form Gallery Table To choose the required mode activate the relevant button on the right hand side of the Image Database window Loading of images into the Image Display window is possible in every display mode The buttons on the right have the following functions Form button Show image database in form display mode Form mmml Gallery button Show image database in gallery display mode Gallery ut Table button Show image database in table display mode Table 03 06 B 45 0019 e 4 19 Carl Zeiss Load button Load Subset button tb on cr pt T Reuse button ma a i ws iD Refresh button mn T m ta Copy button mi Cl 5 Paste button im a mT ee a Filter button On Filter button RX Filter j Delete button Dele T OPERATION LSM 5 LIVE File Menu LSM 5 LIVE DuoScan Load image and parameter from image database to Image Display window Load image and pa
241. cusing device work range 3 Since the piezo objective focusing device moves from bottom to top during the creation of the Z Stack top and bottom of the Axiovert 200 M have been switched round Z Settings ee e Bidirectional Z Scan in time series 4D SS zs 20 2 Der Mark Frbi Hypalre 2 Sedii mses a a Interval 1 ___ ari SeanDiecion cz o aN F Levsina kepre Keep Sic T Lo u e To compensate possible image deviations in the Z direction in bidirectional focus mode use the ii Similar effects can occur when using the fast piezo Z drive in Bidirectional mode with multiple stacks Like for X Y scanning there is a uni and a bidirec tional mode here too Fig 4 68 Corr Z Slider Corr Z slider by typing in the deviation occurring in the adjacent image stacks of a time series B gt If only one time point stack is acquired the Corr Z slider has no effect 4 84 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Auto Z Corr The function Auto Z Correction allows a linear variation of Detector Gain Ampl Offset and Ampl Gain values between the different slices of a stack e Click on the Auto Z button the Auto Z Auto Z Brightness Correction Brightness Correction panel opens Set Reference set 4 Set B The buttons Set A and Set B permit definition of two distinct gain offset settings at two different Z PE t Move Move
242. d Light button The shutter is switched on and off When using the X Cite 120 additional controls are available See below for description Reflector button Push and click reflector cube can be selected via graphical pop up menu Objective button Objective can be selected via graphical pop up menu Condensor button Numerical aperture of the condensor is set via input box or slider Turret position selected from graphical pop up menu only for motorized condensors By clicking on the Close button the value is stored and the Condensor frame is closed Field Stop button Opening of luminous field diaphragm transmitted and reflected light can be set via input box or slider By clicking on the Close button the value is stored and the Field Stop frame is closed Filter button Transmission values for attenuation filter transmitted and reflected light is set via input box or slider for the front or rear filter wheel in accordance with the available filter steps By clicking on the Close button the value is stored and the Filter frame is closed Aperture Diaphragm Diaphragm opening is set via input box or slider By clicking on the Close button button the value is stored and the Aperture Diaphragm frame is closed Transmitted Light button Transmitted light is switched on off via ON button in the Transmitted Light frame setting of light intensity can be varied via input box or slider 3200 K color temperature for photo documentation can be switch
243. d also deleted HEME Fig 4 15 Filter window Edit panel The following features can be used as filter functions under Field Name Words or row of letters from the name of the image Description Words or row of letters from the description of the image Scan Mode Scan Mode in which the image was created Stack or Plane Date Time Date Time of image acquisition Planes z Pixel size of the image in the Z direction e g 10 Lines y Pixel size of the image in the Y direction e g 512 Samples x Pixel size of the image in the X direction e g 512 Z Step Distance of Z Slices in a Z Stack in um User Name of the user as entered in the WINDOWS NT login Time series Selection of time series e Open the Field list box and select the filter feature e g Scan Mode 4 26 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss The following operator signs can be activated under Operator equals gt larger lt smaller larger equals lt smaller equals lt gt smaller larger e Select the relevant operator sign e g from the Operator list box The relevant value or a combination of characters for the filter function Field is entered under Value via the keyboard e Enter the relevant text or value e g Stack e Click on Add The defined filter feature is displayed in the work box of the Edit panel and is therefore activated e g Scan Mode stack If a furth
244. d only within a rectangle which is defined by the greatest extension in X and Y of all the individual figures together i e the pixel number and the data quantity of the Image Display window are reduced s 4 92 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss e In the toolbar of the Interactive ROI Definition panel click on the symbol of the area you want to use to mark the region of interest in the scanning image Five different area symbols are available in the form of buttons e Click on the marking area and keep the mouse button pressed to drag the area into the region of interest in the scanning image The marking area will be numbered automatically and entered in the Interactive ROI Definition panel with its position and dimension parameters and the appropriate number e The dragged marking area is marked by clicking on its outline its size can be changed by clicking on the marking points Clicking on the area edge beside the marking points allows repositioning of the area on the scanning image irs The digits of the ROIs can be shifted independently of the contours of the figure e f you have framed all the required ROIs in Add ROI List x accordance with steps 2 to 4 you can store these ROIs under any required name via the Add to Lists button e The Add ROI List window will appear Enter any required name to store the ROIs and click on the OK button BOI list Hame Image 4 C
245. db Eal Create type Database Files mdb Cancel Fig 6 Create New Database window Fig 7 Create New Database window Turning on the lasers g m Click on the Acquire button in the Main menu to open the Acquire subordinate toolbar Maintain Fig 8 Acquire subordinate toolbar 03 06 5 A e Click on the Laser button to open the Laser Control window Laser 7 Laser Control e Select the appropriate Laser Unit by clicking on the name ot it Wavelength a i l l Toptica 405 50mw 405 nm Jr e Click on the On button to switch required p Moo J is laser s to on or standby if required Sapphire 488 468 nm Toptica 405 Kirni Maximum Power 50 0 mw Wavelength 405 nm Status Ready Fig 9 Laser Control window Setting the microscope Changing between direct observation or laser scanning The VIS TV and LSM buttons switch the beam path and indicate which beam path has been set in the binocular tube of the microscope Click on the VIS button to set the microscope for direct observation via the eyepieces of the binocular tube lasers are off lt S VIS a e Click on the TV button to set the microscope camera observation if connected via camera m adapter of the binocular tube A e Click on the LSM button to set the microscope screen observation via laser excitation using the LSM 5 LIVE and software evaluation Setting the microscope and storing the settings e Clic
246. dinate toolbar of the main menu The Kinetic Analysis window opens e Click on the Close button 4 146 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Process Menu Carl Zeiss 4 6 17 2 Function Description Click into window Channel pull down menu Kinetic Model pull down menu Background Region checkbox Reference Region checkbox Combine Regions checkbox Apply 03 06 Allows to select the image series of the FRAP experiment to be analyzed by clicking into the appropriate image window Allows to select single channels or all channels for analysis Allows to select the mathematical model mono or double exponential model for fitting the data Displays a button Select Click that allows to chose the region of interest which represents the mean background intensity that should be used to correct the data First Click Select Click than click into the ROI that has been drawn using the drawing tools in the Mean ROI image display If a region has been selected a cross appears next to the button Displays a button Select Click that allows to chose the region of interest which represents the fluorescence intensity of a reference cell which has not been bleached The mean intensity within that region is used to correct the data at each time point for any bleaching artifact that occurred during the imaging process First Click Select Click than click into the ROI that has been drawn using the dra
247. dow Info display 03 06 B 45 0019 e 4 311 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 27 Additional Display Mode in Time Series 4 13 27 1 Display Mean This function allows to display the intensity time diagram mean intensity in user defined ROIs over time display the intensity time diagram for volumes 3D ROIs within a Z Stack over time use frame time series and frame Z Stack time series as input show the intensity values in table form and copy table to clipboard or save as text file show separate diagrams for each channel in a multi channel image The settings of Chan Zoom Slice Contr and Palette apply Click on Mean will display the Mean of ROIs toolbar Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed To use a similar functionality while scanning use the optional Mean of ROI function with the Time series control e Click on the Mean ROI button 4 312 The Mean of ROIs image display toolbar will be displayed on the right The used ROIs become visible in the image and the Intensity Time diagram is shown on the left of the Image Display window Unname d16 AIM Select Display Mean of RAOls ing scan o Type Position Dimension TAR M1 Ee 43 152 6 OO ef o Diagram 1 Chan ROI f Mono Area f Mean Threshold Chi Ch3 Ch4 E
248. dure green for ready and red for busy Store current Position Storage of the current aperture setting button Move to stored Position Aperture setting is reset to the position last stored button Adjust Automatically Automatic aperture adjustment button Fast Adjust mode check If this check box is activated the aperture adjustment is only performed in a box limited area Used for readjustment Pinhole LIVE_PH2 Description LIVE Pinhole Che Diameter um 71 Ss gt Pixel Shitt 544 _ gt _ Position Y hdd KI lt S i gt Shore Current y Move To Stored l T m TA i Adjust uel e menl Automatically T Fast Adjust Mode Fig 4 228 Pinhole panel 4 216 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Maintain Menu Carl Zeiss 4 10 3 3 Collimator panel Collimator Description field Positions field Store Current Position button Move To Stored Position button Move to Optimal Position button 03 06 Selection of the collimator IR VIS or UV VIS to be adjusted via Collimator selection box display of selected collimator in the Description field Setting of collimator position using the slider or arrow buttons the display to the right of the slider indicates the current position status display for setting procedure green for ready and red for busy Stores the current collimator position Sets the collimator to the stored value Starts the automat
249. during operation and which might cause damage to the unit The NOTE symbol will help you to optimally solve your work problem It represents a practical tio which will help you to find out which settings and methods are capable of improving or accelerating a procedure The HOT SURFACE symbol warns against hazards for the user that might arise when touching the lamp housing during operation The MAINS PLUG symbol remembers service personal to pull the mains plug before opening the device housing Depending on the problem these operating instructions will supply you with various possibilities e f you want to know where to find certain general areas of information refer to the following outline of sections to get a general overview e You will find a detailed table of contents at the start of every chapter There you will see at a glance what topics are dealt with in detail Always remember The time you invest in getting acquainted with the product will pay 03 06 for itself many times over in your application task B 45 0019 e lI Carl Zeiss INTRODUCTION LSM 5 LIVE LSM 5 LIVE DuoScan Contents 10 Notes on Device Safety This section contains general notes on device safety safe operation and possible hazards caused by failure to observe the instructions LSM 5 LIVE Setup Requirements The Setup Requirements section outlines the installation and supply requirements of the LSM 5 LIVE Microscope Systems together with
250. dvantage faster image acquisition Sere WA i BF 415 480 E m Disadvantage cross talk between channels Chi2 Contig Multi Track Z Ate Use for double and triple labeling sequential scanning line by line or frame by frame aan VA Advantage when one track is active only one detector and one laser is switched on This reduces cross talk Disadvantage slower image acquisition Q iis e Click on the Config button in the ames Acquire subordinate toolbar to open the Configuration Control window Specimen Fig 12 Configuration Control window for Single Track The Configuration Control window appears Fig 12 Setting for single track configuration in Channel Mode e Select Channel Mode if necessary Fig 12 e Click on the Single Track button in the Configuration Control window Fig 12 The Beam Path and Channel Assignment panel of the Configuration Control window displays the selected track configuration which is used for the scan procedure e You can change the settings of this panel using the following function elements Activation deactivation of the excitation wavelengths check box and setting of Excitation excitation intensities slider Open the Laser Control window via the Laser button Selection of the secondary dichroic beam splitter NFT position through selection from the relevant list box Selection of an emission filter through selection from the relevant list box r
251. e Resizing Click on the handle and hold down the mouse button drag the handle release the mouse button Movement Click on the line and hold down the mouse button move the entire intersection line release the mouse button Line with arrow button open arrow Creation of the intersection line to define the position of the profile to be produced in the image Click and hold the mouse button drag the line in any required direction release the mouse button to end the procedure The profile diagram changes online Line button This button allows you to determine the line thickness of the intersection line Measure button Activates the Profile measurement mode in the profile diagram The required tools are displayed to the right of the profile diagram see Profile measurement mode page 4 294 z x 1 button Sets the z x ratio in the profile diagram to the value 1 Check the following creation of a circle using the relevant tool really results in a circle in the profile display Measured angle values correspond to the actual slope of the line displayed Color button Clicking on the Color button opens a color selection box in which the color for the intersection line can be selected with a click of the mouse B 45 0019 e 4 295 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Profile Activating the Profile measurement mode If you click on the FL button the Profile window with the tools of th
252. e Start Cancel Edit on stop OPERATION LSM 5 LIVE Macro Menu LSM 5 LIVE DuoScan 4 8 2 Macro Control 4 8 2 1 Open Close the Macro Control Window e Click on the Macro button in the Macro subordinate toolbar of the Main menu Saves Unload This opens the Macro Control window e Click on the Close button to quit the window Delete Editor Info 4 8 2 2 Edit Macro Function Recording This function allows you to manage project data Macros can be recorded stored performed edited Stop and if required deleted e Press the Edit Macro button to switch to the Macro and Recording panels Fig 4 169 Macro Control window 1 Macros panel New button Load button Save button Save As button Unload button Edit button Run button Step button Delete button Editor button 4 168 e Click on the Close button to quit the window Creates a new project Opens an existing project Stores the project on the hard disk Stores an existing project under a new name Removes the selected macro trom the Macros list Allows macros to be edited and debugged The editor Microsoft Visual Basic is automatically located at the beginning of the relevant macro Runs a macro Opens the editor for line by line editing debugging Deletes the selected macro Opens the editor Displays the processed area of the macro edited last B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoS
253. e corresponding connections on the microscopes are shown in Fig 1 16 2 Fig 1 19 1 and Fig 1 20 1 Remove the connection of the microscope no longer in use at the electronics rack and plug in the connection of the microscope which should be used instead Fig 1 19 CAN connections on the Axiovert 200 M 1 One of the plugs is occupied with the connection to the electronics 1 24 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan User Interface 3 After the connections of the non used microscope are disconnected and the ones of the used microscope are connected the system can be switched on again Before initializing the system with the LSM Software make sure to use the right database according to the microscope in use It can be chosen via the icon Stand Select 03 06 B 45 0019 e Fig 1 20 Carl Zeiss The Axioskop 2 FS MoT is connected to the electronics via the Interface Control The CAN connection is fixed 1 to the control box 1 25 LSM 5 LIVE LSM 5 LIVE SETUP REQUIREMENTS Contents LSM 5 LIVE DuoScan Carl Zeiss CHAPTER 2 LSM 5 LIVE LSM 5 LIVE DuoScan SETUP REQUIREMENTS CONTENTS Page 2 LSM 5 LIVE SETUP REQUIREMENTS ccccsscececcsseeeeseeneeeseeeneeeseeasneesseaneessenseeessones 2 2 2 1 Genera RANA oa a A E 2 2 2 2 System Components and Recommended SetUp s essisessirerrirerrrisrrirerrrrerirerrrrrrrrrerrrrerrn 2 2 2 3 POON CUI SIEN aae E aauntet
254. e individual setting will be retained when the database is closed 4 9 2 5 Database Gallery Viewer The Database Gallery Viewer tab permits the image information to be displayed in the Gallery mode of the database to be activated by clicking on the relevant check box 4 9 2 6 Import Export Use separate path for Import or Export This option permits the change of the path setting for use of the Import or Export function File menu 1 Save most recently used path at exit and reuse at next program start On activation of this option the path used last is automatically selected again in the Import Images or Export Images and Data window 2 Use the following path at program start OPERATION Options Menu Carl Zeiss Poreseneeseessasensesenseenenesssessssssssssssesssssssccsessassess Autosave Database General Database Table Viewer Database Gallery Viewer lenesssescesssesesssssscscccsssssssacssasasaceseesssesesseesessed T Table columns to display M Name MV Pixel Depth M Objective Processing Summary Description V Stack Size Pixel T Beam Splitters Processed Size Notes M Stack Size um D Wavelength P Image Pathname M Date Time Position Filters User M ScanMode M Scaling Pinhole Automatic column width calculation Save and use interactive column width setting Database Table Viewer tab Fig 4 204 Autosave Database General m Image information to display M N
255. e objectives working distance a warning can be displayed 4 8 9 3 Editing a Macro h l m Beam Path Following Example shows a macro which acquires five subsequent images and moves the stage 10 microns in X and Y following each image The T can Parameters m images are stored and numbered in ascending order EF C E Image Display T pa a Move eee mear to Database 22signments e Arrange and connect the action blocks Fig 4 191 43 ds nc vesh T Fig 4 191 Arrangement of action blocks e Assign the value 1 to a variable named Index Property alue Fig 4 192 Index Fig 4 192 Assignment of the variable 03 06 B 45 0019 e 4 195 Carl Zeiss OPERATION Macro Menu LSM 5 LIVE LSM 5 LIVE DuoScan e Click the single action blocks and assign the values to the listed properties beam path and scan parameters can be chosen according to the sample Use Property Value LOM Pixels 8 LOM Pixels Y LSM Step LSM Zoom LSM Rotation 7 LOM Offset pm LOM Offset pm LSM Pixel Period ps Camera Mode Camera Pixels 2 Camera Pixels r Camera Offset Pixels Camera Offset r Pixels Pirels Z Scaling Z m Offset 2 ur Bits per Sample Fig 4 194 Al Al 1 600000 45 000000 0 000000 0 000000 2 560000 Mono 1300 s 1030 1300 1030 0 000000 0 000000 1 000000 0 000000 8 bit Property list of the Scan Parameter
256. e position within the Image Display window in pixels and greatest dimension in X and Y in pixels The origin of the position indication lies in the left top corner of the Image Display window OPERATION Acquire Menu Carl Zeiss Fig 4 75 ROI Lists panel Interactive ROI Definition Type Center Position Dimension i A Y Ei Iw 1 Rectangle 176 IE IE 133 Wa Bezier as E 145 lira Iv 3 Folyline 350 372 2s faz W4 Ellipse 103 371 148 E ts OlOO O Y e T Fit Frame Size to Bounding Rectangle of all ROls Fig 4 76 Interactive ROI Definition panel 03 06 B 45 0019 e 4 91 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Check box e g 1 4 Clicking on this check box allows a ROI to be deactivated The tick disappears from the check box as does the relevant marked area trom the scanning image Clicking on the check box again will reactivate the ROI Arrow button Activation of the mouse button to change the size or move the ROIs in the Image Display window Rectangle button Draw of a rectangle in the Image Display window click and keep mouse button pressed drag the rectangle in any direction let go off the mouse button to end the procedure Bezier button Draw of a bezier figure in the Image Display window first click sets the starting point each additional click adds a line double click on the starting point closes the figure and ends the procedure Ellipse button Dr
257. e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION 3D View Menu Carl Zeiss 2 Transparency tab Mode Maximum Mode Transparent Mode Keep Maximum Threshold Ramp Maximum Opacity Brightness 03 06 The color is defined by the z position of the brightness value The transparent projection is built up from the rear to the front The color is defined by the z position at which the original was last higher than or equal to Threshold Activating this option modifies the specification governing calculation of the projection Pixel value at which the ramp rises variable from O to 100 Slope of the ramp variable from O to 100 0 corresponds to a vertical rise Degree of visibility at the top corner of the ramp variable from O to 100 O corresponds to the bottom edge in the diagram The image can be brightened again by modifying the value from 0 2 to 5 Projection Transparency Mode C Masimum Transparent Keep Masimum Threshold po RE Bamp m E Maximum Opacity 50 Brightness o s pe Fig 4 157 Transparency tab B 45 0019 e 4 161 OPERATION LSM 5 LIVE Carl Zeiss 3D View Menu LSM 5 LIVE DuoScan IM Preview Slice i Fig 4 158 Preview panel Fiki SUS on S02 3 ehr F be Fig 4 159 Stereoscopic image red green Finnia ee Fig 4 160 Stereoscopic image split 4 7 3 4 Preview Panel The Preview function permits you to regard the influ
258. e Edit Bleach button in the Acquire Subordinate toolbar of the Main menu The Bleach Control Help190 window appears on the screen e Click on the Close button to close the Bleach Control window The following functions are available on the right hand side of the Bleach Control window The Bleach Control window is closed Close button Bleach button Starts the bleaching procedure Stop button Ends the bleaching procedure B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 8 2 The Settingsj Help191 panel allows you to determine when where and how the bleaching process shall be done only works in connection with time series Furthermore all deleted in this panel Bleach after number scans Scan Number Bleach repeat after number scans Scan Number Different Z Position Different Scan Speed Different XY Spot Bleach Position Settings Panel the settings of the Bleach Control window can be stored reactivated or OPERATION Acquire Menu Carl Zeiss FRAP Myocandiac CX 43 MUT Apply Store Delete Bleach after number scans M Scan Number 5 Bleach repeat after number scare Iw Scan Number i 0 Different Position MW 11 33 pm Momsen m e afr Fl a Fisel Time 6 40 ps Trigger in Hone Trigger out Hone Fig 4 97 Settings panel If this check box is ticked b the bleaching procedure is automatically performed in combination with a time series
259. e ROI or scatter region Colocalization coefficients Weighted colocalization coefficients Overlap coefficient after Manders Correlation coefficients R and R Colocalization coefficients pixels cr coloc _ PIXELS Cy cotoc 5 C5 PIXELS cn toral pixels o 2 total Relative number of colocalizing pixels in channel 1 or 2 respectively as compared to the total number of pixels above threshold Value range 0 1 0 no colocalization 1 all pixels colocalize All pixels above background count irrespective of their intensity Weighted colocalization coefficients 2 Ch boi gt CH 3 i j gt CH se K j SCi Sum of intensities of colocalizing pixels in channel 1 or 2 respectively as compared to the overall sum of pixel intensities above threshold and in this channel Value range 0 1 0 no colocalization 1 all pixels colocalize Bright pixels contribute more than faint pixels Correlation coefficient Pearson s correlation coefficient S Chl Chl pe Ch2 Ch2 vey X Chl Chl gt Ch2 Ch2 Provides information on the intensity distribution within the colocalizing region Value range 1 to 1 1 41 all pixels are found on straight line in the scatter diagram O pixels in scattergram distribute in a cloud with no preferential direction 4 200 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and A
260. e applied immediately A change of the delay during a Time Series is displayed in the Image Display window if the Gallery button Display toolbar is activated fo Apply Store Delete Desciption Trigger in Trigger out None f None None f None None x None x Fig 4 87 Marker panel Function description Marker list box Apply button Store button Delete button 4 100 4 5 6 5 Marker Panel The setting of a marker permits information about the moment in the current time series and any required comment to be assigned to the current scan The time indication is set automatically while comments must be defined before The markers red squares are visible in the Image Display window if the Gallery button Display toolbar is activated On storage of the image all the markers including the time indication and the comments are stored along with the image contents List of the stored combinations of markers Application of the marker combinations selected from the list box Storage of a combination of markers Deletion of a combination of markers from the Marker list box B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Set 1 7 button Setting of a marker during the scan procedure Description Entry of the comments for the marker input box 1 7 Trigger in Selection of the trigger key 1 4 with which the marker is to be set list box 1 7 Trigger
261. e button to see an intensity graph superimposed on the image 03 06 B 45 0019 e 4 261 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan D Convallaria Aull Marker 1 Marker 2 Free selectable line for intensity profile r Apter L Hatay AT B Ligh res i H Ai pri i bat ih jami i ETE riari i i 1 i i riari i i r i ipl lij i Profle rier sa s iE IS cA i Fy Pa LE 1 i re I 4 a A 1 l D i i ta 7 hil F Read Sis S20 20 Dohaneh 0 be Fig 4 266 Image Display window Profile display l aj Select Display Profile Convallaria2 AlM gt Pai i ai N P Y r od y a nA a wt p P P A ow Fa Fa Pi Y Pa vo Pi 7 af a y Pi p t yf a 5 a yf 4 A y g a A i a A A g Pall yy P Pa va a A F Pa jA Pa y aw 5 va ag al 4 A A A A NA X T ny III Sez E i E CORRE TER 1E TET E Diagr in Image Marker Marker 2 Difference 68 86 pum 34 43 pm e T E e a a O o d D f Profile Intensity 250 200 150 100 50 0 Chi Ch2 Ch3 Ready 512 x 512 x 20 3 channels 8 bit Fig 4 267 Image Display window Profile display 4 262 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 21 2 Function Elements The Profile toolbar contains the following function elements SI
262. e cells under investigation in situ or in solutions in vitro Calibration parameters may be saved and reloaded cal 4 6 12 8 Options for Calibration Image Selection Equation or Titration Calibration e Click into image window e Select source channel s e Optional background subtraction e Optional calculation of parameters from overlay region s 4 6 13 Image Scaling The use of this function permits the scaling of a imported image in voxel dimensions Setting the values will provide the voxel dimensions in the X Y and Z directions Z direction not for 2D images 4 6 14 Copy Window Contents The use of this function permits to duplicate the displayed image 03 06 B 45 0019 e 4 145 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 15 Copy Full Resolution The use of this function permits to display the current image with full resolution in a new Image Display window 4 6 16 Paste Bitmap This function allows to paste bitmap images trom the clipboard into a Image Display window Images can be incorporated from other programs such as MS Excel MS Powerpoint or MS Word 4 6 17 Kinetic The use of this function option permits a correction of FRAP data for bleaching and background the fitting of the FRAP data to a mono exponential or double exponential model for intensity recovery 4 6 17 1 Open Close the Kinetic Analysis window e Click on the Kinetic button in the Process subor
263. e center The Scan Mean of ROIs toolbar with further function elements is additionally displayed on the right hand side The major purpose of these function elements is to vary the display of the recorded Mean ROI By selecting the appropriate options see Options menu Settings Scan Mean of ROIs you can activate the following additional functions Display of the live image in the Image Display window of the Mean ROI function used ROIs only Scan of the complete image if Live Image has been activated Saving of the complete time series if Live Image has been activated 4 106 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss The following functions are available Type Position Dimension a Y A f C 176 136 135 133 2 362 407 102 102 3 X 381 182 145 174 lt 1 41 41 Diagram t t Z Ex E fo 1 Chan Aol Mora Scaling Automatic Time Range 10 L Number Cycles Image Table Show mage Show T able Save Table 03 06 Display of the data of the ROIs used for the creation of the MeanROIl identical to the Edit ROI window If the check box of a ROI is deactivated the ROI s intensity values are no longer displayed in the Intensity Time diagram 1 button Intensity values for ROI and Channels are displayed in a diagram Chan button Intensity values are displayed separately for each channel used ROI button Intensity values are displa
264. e destination image Fig 4 115 Operators window Copy Ed Click into window Channel New 7 New Image 8 bit Fig 4 116 Copy window 4 127 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 5 2 Performance of the Copy Function e Select Source Channel and Destination and then click on the Apply button The image of the copied channel is then displayed in a new window or in the Image Display window activated for it e The new image can be stored via the Save As function L For Z Stacks or Time Series the entire series of the selected channel is copied 4 6 6 Duplication Image This function permits images including Z Stacks and Time Series to be duplicated completely e f several images have been opened select the image to be duplicated e Click on the Dup button in the Process subordinate toolbar of the Main menu The selected image is duplicated and displayed in a new Image Display window e Use the Save As function to store the image under a new name 4 128 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss 4 6 7 Filter W Filter The filter function permits the subsequent a processing of scanned images via the integrated es Lowpass Sharpness and Median filters Furthermore User defined filters can be installed by the user User defined filters can be stored reloaded and removed Channel Chi X Convallaria
265. e diagram is displayed in the image along the drawn profile line Marker 1 button red Activates the red marker for movement in the profile diagram the marker shown as a red line in the diagram can now be moved to the right or left of the diagram using the mouse The marker in the image display red circle follows accordingly Marker 2 button blue Activates the blue marker for movement in the profile diagram the marker shown as a blue line in the diagram can now be moved to the right or left of the diagram using the mouse The marker in the image display blue circle follows accordingly B 45 0019 e 4 203 Carl Zeiss E E FF e T J C SE ay 2E un ia Tm gu 4 264 BE EE T OPERATION LSM 5 LIVE Display and Analysis of Images LSM 5 LIVE DuoScan Zoom button Zoom function for profile diagram Click and drag a rectangle over the area to be enlarged in the profile diagram the selected area is enlarged on release of the mouse button The zoom function can be performed several times A click with the right mouse button will reset the original size Reset Zoom button Resets the zoom factor of the profile diagram to the original size Show Table button The profile diagram is displayed as a table at the bottom of the Image Display window Copy Table button The profile table is copied to the clipboard Save Table button The profile table can be stored as a text file extensi
266. e displayed and further marks can be added at spots of special interest by a mouse click in the Tile Scan image By activating the Move To button the stage can be moved to the individual marks set in Tile Scan in the same way as it is moved to the marks set in the Stage Position panel Creating an overview image e Set the number of tiles for the frame in the Tiles Numbers X Y input boxes of the Tile Scan window The resulting frame size is displayed on line e Click on Start The overview frame is scanned and displayed on the screen in a new Image Display window 4 118 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Tile Scan AlM Ready 2046 1536 3 channels 8 bit Raw image data Display Zoom 14 Palette No Palette Fig 4 105 Image Display window of a Tile Scan e Activate the Move To button e Inthe tile scan image move the target to the required spot of the frame dragging with the mouse The microscope stage then travels to the selected position Or e Activate the Mark button e Seta mark at the spot of interest by clicking with the mouse in the Tile scan image A cross with the consecutive number of the mark is displayed in the Tile Scan image The new mark is also displayed in the specimen carrier Stage Position panel and included in the Marks selection box e Select the mark in the Marks selection box and click on the Move To button in the Stage Posi
267. e for the macro to be created in the Rec Name input box of the Recording panel Start button Starts recording Cancel button Cancels the recording procedure Stop button Stops recording Edit On Stop On stopping the recording procedure the macro editor is automatically opened at the relevant position Proceed as follows to record a macro e Enter a name for the macro to be created under Rec Name in the Recording panel e Click on the Start button to start recording the macro e Then perform the operations to be stored e g Click on the Find button in the Scan Control window A Find scan will be performed Click on the New button in the Scan Control window A new Image Display window will be opened Click on the Single button in the Scan Control window A Single scan will be performed e Then click on the Stop button to end the recording Cancel enables you to cancel recording If recording was successful the entered Rec Name will then also be available in the Macros list box of the Macro panel The new macro is automatically assigned to the current project It is possible to assign as many macros as required to a project e Click on the Save button to store the new macro Rec Name Start Cancel Stop Edit On E Stop Fig 4 172 Recording panel 4 170 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss Proceed as follows to perform a macro e Select the required m
268. e overlay element or the scatter diagram with the color selected under Mask M Closed free shape curve button Creation of a closed Bezier figure in the scatter diagram The first click sets the starting point each additional click adds a further line a click with the right mouse button closes the figure and ends the procedure O Circle button Creation of a circle in the scatter diagram Clicking and holding down the mouse button sets the center point drag the diameter and release the mouse button to end the procedure O Color selection button The colors displayed in the selection box can be assigned to the mask elements with a click of the mouse The currently selected color is always displayed in the larger rectangle top left of the selection box E Clear Mask button Removes the color filling from an overlay element or from the scatter diagram P The function can be activated by clicking on one of the geometry buttons e g 27 polyline e The figure of interest can be marked in the image by cursor control in conjunction with a mouse click e f more than one Region is drawn either the mean histogram values for all regions is displayed or only the values of the activated region if it is marked by clicking on the drawing line with the mouse e Areas can also be excluded from the analysis By clicking on the Flood fill button paint jar and moving the cursor to the area to be excluded causes the remaining area to be
269. e protile measurement mode appears This window can be moved as required over the entire screen Fig 4 296 Tools of the Profile measurement mode Tools of the Profile measurement mode The tools of the Profile measurement mode have the following functions A Zoom button Zooming of a section of the profile diagram Click and drag a rectangle over the area to be enlarged in the profile diagram release the mouse button to enlarge the selected area The zoom function can be performed several times A click with the right mouse button resizes the profile Marker button Activation of the marker functions for the intersection line The red and blue marker lines in the profile diagram can now be moved using the mouse After movement of a marker line in the profile diagram the relevant marker red or blue circle follows along the intersection line in the 2D and Iso Lines mode k Arrow selection button Activation of the mouse button for selection resizing or movement of one of the following drawing elements in the profile diagram Resizing Click on the handle and hold down the mouse button move the handle release the mouse button Movement Click on the line and hold down the mouse button move the entire drawing element release the mouse button Inclined Line button Creation of a straight line in the profile diagram Display of distance inclination angle dxdy and dz Click and hold down the mouse button drag the line i
270. e relevant data points 10 11 and I delta which are calculated performing the Kinetic Analysis 03 06 B 45 0019 e 4 151 OPERATION 3D View Menu Carl Zeiss 4 7 3D View Menu LSM 5 LIVE LSM 5 LIVE DuoScan The 3D View functions serve to record and play back series of images for 3D display of microscopic structures e Inthe Main menu toolbar click on 3D View This opens another subordinate toolbar in the Main menu A LSH 5 LIYE Ei Ei File Acquire Process 3D View Macro Options Maintain Window Help Fa Process fi Fig 4 144 3D View menu Depth Coding Click into window c ce cn Depth Coding Depth Coding Transparency Mode Front View C Rear View Threshold Contrast o p Brightness j5 Z Display IV Scale Bar T Grey level D Preview Fig 4 145 Depth Coding window Maintain 4 7 1 3D DepthCod Color Coded Depth Map By means of the Depth Coding function the depth information contained in a sequence can be colored with the colors of the rainbow in which case blue stands for front and red stands for rear A stack of images must be available 4 7 1 1 Open Close the Depth Coding Window e Click on the DepthCod button in the 3D View subordinate toolbar of the Main menu This opens the Depth Coding window e Click on the Close button to quit the window 4 152 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE
271. e same resolution as the Input sequence A change will be needed if image sequences with different resolutions should be combined Rising the grey value range to 12 bit will not enhance the display quality or measurement accuracy The smooth and morphology functions will produce results with finer gradations If Clip Grey Values is selected the output grey values are clipped to the Low L and High H values If Clip Grey Values is not selected output grey values beyond the Low and High value range are possible 03 06 B 45 0019 e 6 19 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan The Output histogram shows the resulting histogram The horizontal axis represents the grey values from O to the maximum which is either 255 or 4095 depending whether the input is 8 bit or 12 bit The vertical axis represents the pixel count The selected range is marked by the borderlines in the histogram The blue line or L indicates the lower boundary the red line or H the upper one C indicates the center of the range There are three ways to change the range clicking and dragging the borderlines with the mouse Entering a new value in the appropriate text boxes clicking on the buttons 2 or using the arrow keys from the keyboard To alter the values within the histogram move the mouse pointer over one of the three coloured lines until the shape changes Press and hold the left mouse button to move the line to a new position To change the val
272. e scan e g X 512 Y 512 scan e Enter a Exposure Time of 5 to 10 frames per second for example to start with e Start with the following settings on the Pixel Depth Scan Direction amp Scan Average panel Data depth 8 bits Scan direction J unidirectional Average Number 1 e On the Zoom Rotation amp Offset panel set a Zoom of 1 L Using the Fast XY button is a convenient way of creating an overview scan Fig 4 307 03 06 B 45 0019 e Image Optimization 74 Scan Control Sethe De Objective Lens Image Size amp Line Step Factor Plan Mecfluar 104 013 Objective Frame Size 128 256 Al2 1024 512 a 512 512 x512 Exposure Time a oo fr 10 3 Frames per Second Pixel Depth Scan Direction amp Scan Average Data Depth Bit 12 Bit Mode Frame Method Mean Scan Direction a r Rim biet 1 2046 Format Zoom Rotation amp Offset Offset Offset Offset y 0 00 um 0 00 um Scan Control window Mode Carl Zeiss Single 4 317 OPERATION LSM 5 LIVE Carl Zeiss Image Optimization LSM 5 LIVE DuoScan 74 Scan Control Channels Pinhole 102 W gt 1 Mas Optical slice lt 13 6 um Pinhole amp 0 97 Ain Equival Detector Gain 1 S gt AmpliierOftset 0 Ado H a Time 0 09 Al mie gt 10 3 Frames per Second Line active Transmission 2 B A C asan
273. e sequence is preserved and or deleted in the Output image sequence Only one channel of a multichannel sequence can be selected as Input Output will always be a single channel sequence The vertical scaling of the histogram can be adjusted with the scroll bar at the right edge of the histogram This setting has no influence on the function The thresholds Low and High are determined either by moving the borderlines in the grey value histogram or by the scroll bars underneath Furthermore the values for Low Center and High can be set through entry in the corresponding fields To move the lower L and upper H thresholds at the same time move the vertical line in the grey value histogram or set the scroll bar C The Green and Blue Red option buttons of the parameter Colour determine whether the voxels within Green or outside Blue Red of the grey value interval L H are displayed with the corresponding colour If Green is selected the voxels within the selected interval are highlighted in green The rest of the image retains its original grey values The voxels with the grey values Low and Low 1 are displayed in blue The voxels with the grey values High and High 1 are displayed in red 03 06 B 45 0019 e 6 33 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan If Blue Red is selected the voxels with grey values within the interval Low High remain unchanged Voxels with grey values less than Low are highlighted in bl
274. e stand is not allowed be located in the beam path because of the laser light already is polarized c On the illuminator support as an option there is mounted a LSM 5 software controlled switching mirror fully motorized Alternatively the light is directed to the LSM 5 T light detector or enables conventional transmitted light observation d The focusing screen for conventional transmitted light Is located in a support in front of the halogen lamp housing e Further information to halogen lamp and condensers you will find in the Axiovert 200 M operating manual 4 Reflected light fluorescence All Axiovert 200 M fluorescence accessories exceptional the reflector slider can be used Further information you will find in the Axiovert 200 M operation manual 5 Imaging optics Optovar sliders are not usable The analyzer for conventional DIC mode will be operated from the right side and is located just below the nosepiece Use of sliders with auxiliary objects 473704 14 0000 000 is not possible 6 Photo equipment The stand hasn t have an integrated SLR port but microscope cameras as described in the Axiovert 200 M operation manual can be used 7 TV adaptation The TV port aside and the tubes can be used as described in the Axiovert 200 M operation manual The TV interface side port or base port can be used with TV adapters 44 or LSM adapters 03 06 B 45 0019 e 3 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 LI
275. e the side ratio as required Release the mouse button 4 242 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 12 Select Copy This function allows to copy the current displayed image into the clipboard Click on Copy will be immediately effective From the clipboard images can be incorporated into other programs such as MS Excel MS Powerpoint or MS Word To export image series use the Export function in the File menu e Click on the Copy button The content of the Image Display window is copied to the clipboard e Start the clipboard application of WINDOWS e Select Paste in the Edit menu of the Clipboard application 4 13 13 Select Save This function allows to save the image s of the Image Display window into an Image Database by not showing a dialogue and using the automatic assigned and incremented image name and a predefined existing Image Database Prerequisite Autosave is checked in the Settings function with the Autosave tab Click on Save will be immediately effective When the prerequisite is not met the Save As dialogue Is displayed In the Options menu in the function Settings with the Autosave tab parameters such as an automatically incremented filename can be determined and the Autosave activated deactivated 03 06 B 45 0019 e 4 243 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 1
276. e with the configuration specified for the given block Autofocus search parameters Z offset as well as the Z range the distance in the z direction over which the autofocus function searches for the plane of maximum intensity can be set independently for different stage locations on the systems with the motorized stage 4 176 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss 4 8 5 Kollimatic VBA Macro to calibrate the LSM 5 LIVE depending on the current system configuration e g objectives filters Prerequisites System Warm Up at least 2 hours for procedure please read the User s Manual and environmental conditions according to setup requirements OptimizeLIVE lvo Macro ensure both channel sliders set to O Proper adjustment of collimators and Confocal Slit Aperture CSA 1 2 this has to be done by the local Service Representative AOTF RGB V linearized properly with Macro AOTFFitlin lvb All used objectives have been stored in the database according their exact order in the nose piece Necessary calibration slides are available 1x Bead Slide 1um for dry objectives 000000 1371 460 1x Bead Slide 200nm for immersion objectives 000000 1371 461 to be ordered from Carl Zeiss Jena Basic Functions of the Macro The KolliMatic macro features two main parts Basic Calibration and Calibration Matrix A Basic Calibration Needs to be done only once wit
277. ect Slice This function allows to select and view individual slices from a Z Stack or a time series when images where acquired in frame mode 3 The button is grayed when these conditions are not true Click on Slice will display the Slice toolbar Any changes done with this toolbar are effective immediately e Click on the Slice button in the Select toolbar The Slice toolbar is displayed on the right hand side of the Image Display window e lf you click on the Slice button again the Slice toolbar is removed I Donvallarnra Alle Example Slice No 10 from a Z Stack or time series of 20 slices Ready S124 512 20 3 channeds 8 be Fig 4 241 Image Display window Select Slice 03 06 B 45 0019 e 4 229 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 6 Select Overlay This function allows to select from a set of drawing functions such as rectangles and arrows add a scale bar to the image use a set of interactive measurement functions for length angle and size 4 13 6 1 Functional Description The overlay function uses a plane separate from the image plane the graphics plane and does therefore not change the content of the image s The button is only available if the XY or Split XY Display functions are selected Otherwise it is grayed Some of the Display functions such as Ortho or Cut turn the overlay graphics off temporarily Any changes done wi
278. ed accordingly e Keep Interval enables the Interval um slider which allows to vary the interval between the slices The number of slices is matched accordingly the limit of the Z Stack is adjusted e Click on the Start button to start the recording of the Z Stack In case the upper and lower limits of the stack have been switched round automatic matching will be performed by the software since the stage of the Axio Imager Z1 always moves from bottom to top and the nosepiece of the Axiovert 200 M always moves from top to bottom is Setting via Range is not possible via the Mark First Last function i e the lines cannot be shifted The Fast Z Line functions is not available in frame mode When you change from Mark First Last to Z Sectioning or vice versa the values are updated in the Z Sectioning tab 03 06 B 45 0019 e 4 83 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Z Sectioning Mark First Last Hyperfine Z Sectioning Hyperfine Z Sectioning tab Activation of this tab is only possible if the piezo objective focusing device has been connected Hum Slices 19 4 j gt Interval um 0 989 gt Feer Keep Interval Keep Slice Fig 4 67 Hyperfine Z Sectioning tab The piezo objective focusing device can be controlled via software see Stage page 4 114 The accuracy of the piezo objective focusing device regarding the step width in the Z direction lies in the range of 60 nm The p
279. ed is performed with a click of the mouse 4 9 2 8 Image Status Display This tab permits selection of which image information is displayed on opening of an image or on activation of the Info button of the Image Display window Furthermore you can determine which information will be displayed in the Image Status bar On activation of the Show status display upon opening of a new image display check box the image information is automatically displayed immediately after opening of the Image Display window Info button is activated 4 9 2 9 Print Status Display This tab permits selection of which information is displayed in print preview On activation of the Print Status Information check box the status information will be printed 4 204 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 9 2 10 Recording Reuse The parameters to be taken into consideration for the use or load of a recording configuration are set in this tab As an option you can also determine whether the objective setting shall be taken over when the Reuse function is used 4 9 2 11 Time Series In the Time series tab you can determine whether the time for the recording of a time series is set as Cycle Delay or as Time Interval Cycle Delay is the interval between the end of one scan process and the beginning of the next Time Interval is the interval between the beginning of one scan process and the beginning of the next You can select
280. ed on via 3200 K button in the Transmitted Light frame By clicking on the Close button the Transmitted Light frame is closed 03 06 B 45 0019 e 4 41 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 74 Microscope Control Microscope Settings Close Less Field Aperture Fitt Reflected Stop Diaphragm EEN Light i Lo 643 les 100 000 9 Off O04 Objective Plan Meofluar 1020 3 Condensor Aperture 60 06 Transmitted Light Field Stop Filter io Ut 5 100 000 Fig 4 27 Axio Imager Z1 Control window e Click on the desired microscope configuration Recording of microscope settings The upper part of the Axio Imager Control window shows the recording functionality of microscope contigurations Complete microscope configurations can be created and applied The Store button permits existing microscope configurations to be stored under any name The Apply button permits existing stored microscope contigurations to be loaded The Delete button permits existing microscope configurations to be deleted The Assign button permits the assignment of a microscope contiguration to a button Load a microscope configuration An existing microscope configuration can be loaded as follows e Click on the arrow button This opens a list box of all stored microscope contigurations e Browse through the microscope configurations by clicking or use the scroll bar at the side of the
281. ed out f The stands dispose of five additional ports two side ports front ports and base ports The side port or the front is equipped with the LSM 5 special interface one of the others with the TV interface The LSM 5 LIVE scanning module can be mounted to the special interface port Different camera systems can be adapted to the TV interface using the TV adapters 452982 83 92 94 95 97 98 0000 000 2 Specimen stages and fine focus drives a Mechanical stage The stage must be mounted with the coaxial drive on the right side of the stand b Scanning stage c Piezo objective focus drive 3 6 B 45 0019 e 03 06 LSM 5 LIVE INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 LIVE DuoScan Performance Features of the LSM 5 LIVE Carl Zeiss 3 Transmitted light illumination a The illuminator support contains a security circuit which activates a shutter preventing laser light from reaching the stand when the support is moved to back A complementary shutter built in the stand prevents laser light from reaching the eye pieces during scanning mode b The illuminator support is equipped with a rotatable polarizer The Axiovert 200 M description contains the adjustment for DIC mode during conventional observation For scanning transmitted light DIC mode the polarizer in the transmitted light support works like an analyzer and must be adjusted in such a manner that direct laser light will be blocked The conventional analyzer slider in th
282. ed wavelengths for the scanning procedure Display modification of the user accessible program Settings of the LSM 5 software e In the Main menu toolbar click on Options This opens another subordinate toolbar in the Main menu S LSM 5 LIVE Fig 4 199 Options menu 4 198 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Options Menu Carl Zeiss 4 9 1 Dye DB Function The Dye DB function is for information only and permits access to the database contained in the system including a list of suitable dyes for fluorescence microscopy The database contains a comparison of tables of dyes optimum excitation wavelengths and maxima of emission wavelengths e Click on the Dye DB button in the Options subordinate toolbar The Dye database will be opened and displayed on the screen e Click on the Close button to exit the Dye database PFluorochrome Ext fom Ex nm LE fom Em nm 4 Metylumbelliferon E Amino Quinolin T Amino Actinomycin D Acndingelb Acrdinorange D NA Acidinorange A NA Acitlavin Feulgen Alexa 350 Alesa 430 Alexa 485 Alexa 532 Alena 546 Alera 568 Alesa 594 Alizarinkomplexon Allophycocyanin AMCA Arnino Methylourniarir Atebrin Auramin Auraphospin B Phycoerythrin BAD BCECF Berbernsulfat BFP blue shifted 66H BFP2 Blue FluoS pheres BOUBO 1 Fig 4 200 Dye database window 03 06 B 45 0019 e 4 199 OPERATION LSM 5 LIVE Carl Zeiss Options Menu LS
283. eeas 4 24 4 4 3 5 Load Image with Reduced Resolution Subset occ cece eecccceeeceeeeeeeeeeeeeeeeneeeeaneeeeeaneeees 4 25 4 4 3 6 Update Image Database Display Refresh cccccccccccceseeeccecceeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeaaas 4 25 4AT CODY Pase Image s 10 THC LID OOO ict sorters a a a hit etinedss 4 25 4 4 3 8 Display Images with Certain Features Filter 00 cee cccccceccccceeeeceeceseeeeeeeeeeeeeeeeeseeeeeeaneeeeees 4 26 PASST OMRE Gk LO AG ION eaa a eae ern ey Sent N 4 28 4 4 3 10 B LCG WOM A A A tet A onsen O A EEE A IAEE E ATT 4 28 4 4 4 Save an Image to the Image Database cc ccccccccccceecccee ee eeeeee ce eeeeeseeeseeeeaeeeeeeeaeeeeeeanaes 4 29 4 4 5 MOON TION NMA OCS ive deere eno eee tenet ier ik oe alee r ena thet Aan 4 32 4 4 6 EO OE OTIC OS senen aa caceesseteaset ean E an E 4 33 4 4 7 KIUB IE caesar arate ance Match abe ete pees sth dence ecco ak a ena eee Setanta 4 34 A Asda OVE TOODA sintcutata acces tects chord ts a a a E aae aas 4 34 AATA PEMA VOM MIM AG OS essan TA S 4 36 4 4 8 Ue oie ao A A ot cunts tania tet E EE nye ci Avani RE E EENET E ace 4 36 4 5 PROGUIN Men aannaaien E a E EOE 4 37 03 06 B 45 0019 e 4 1 Carl Zeiss 4 5 1 4 5 1 1 4 5 1 2 4 5 1 5 4 5 1 4 4 5 2 4 5 2 1 4 5 2 2 4 5 2 3 4 5 2 4 4 5 2 5 4 5 5 4 5 3 1 4 5 3 2 4 5 3 3 4 5 3 4 4 5 3 5 4 5 3 6 4 5 3 4 5 3 8 4 5 3 9 4 5 4 4 5 4 1 4 5 4 2 4 5 4 3 4 5 4 4 4 5 5 4 5 5 1 4 5 5 2 4 5 6
284. eft mouse button changes the rotation direction and speed of the animation L The fastest animation results can be achieved with the advanced surface rendering mode even without additional graphics cards 03 06 B 45 0019 e 4 271 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 23 2 Transparency Projection The transparency projection generates a transparent 3D reconstructed image The elements for image display zoom 3D rotation home coordinate system scale frame and animation function are identical to those of the Any View function of the shadow projection and are operated in the same way The transparency projections Basic and Advanced are perspective tyoe 3D reconstructions with the Advanced projection permitting the perspective impression being varied between parallel and centric projection by changing the View angle The Advanced projection also offers the possibility of selecting between fast and precise calculation via the Precise Fast slider at the bottom right in the 3D toolbar Of course the precise calculation method is more time consuming gt Convallaria AIM Display y Transparency Basic Advanced Surace Basic Advanced Full Res Appearance CCC gt AIC Ready 51285128 20 3 channels 3 bit Fig 4 274 Image Display window 3D display Transparency projection Advanced 4 272 B 45 0019 e 03 06 LSM 5 LIVE OPER
285. el 1 BP 495 525 SK LP 550 BP 560 675 SK LP 650 SK BP 700 750 Emission BP 415 480 SK filters BP 455 525 channel 2 BP 495 525 SK BP 550 615 7 4 B 45 0019 e 03 06 LSM 5 LIVE ANNEX LSM 5 LIVE DuoScan Detaching Attaching the Scanning Module Carl Zeiss 7 3 Changing Filters in the Scanning Module For optimum investigation of specimens it is useful to employ filter wheels permitting the motor controlled change between different filters for narrow band or broad band detection depending on the wavelength The number of filters is limited by the capacity of the filter wheel The change of the filter wheel as a whole involves complete readjustment Please ask your LSM service engineer to do this exchange in order to maintain your warranty 7 4 Detaching Attaching the Scanning Module from to Microscope Stands Tool needed 3 mm Allen key ms The user can remove the Scanning Module from one microscope and attach it to another within a few minutes Described below is the change over from an Axioskop 2 FS MOT to an Axiovert 200 M in sideport configuration Before the change over shut down the system as described in chapter 4 in order to avoid damage to the system and loss of data e Loosen the three screws 7 1 1 at the Scanning Module 7 1 2 fitted to the Axioskop 2 FS MOT e Cautiously pull Scanning Module off the Axioskop 2 FS MOT stand e Attach Scanning Module to the left sideport of the Axiovert 200 M minding the
286. el buttons in the Channels toolbar e g Ch1 The color selection box will appear e Click on OFF to deactivate the display of the relevant channel fi A newly assigned color or a channel switched off is not taken into consideration during the following scanning procedure since the setting in the Configuration Control window always applies here 4 13 3 3 Switching to Monochrome Image Channel Colors x Display Current Set of Channel Colors Close e Click on the Mono button in the Channels Cit ge toolbar The image will then be displayed in shades of gray exclusively If you click on the button again the channels will be displayed in color again Define Color i If you want to view the channels ius far individually select the split display by ee clicking on Split xy button in the gat 125 Display toolbar Lum 120 4 redfe 4 13 3 4 Defining a New Color Been ri4 Blue 183 E e Click on the Colors button to open the Channel Colors window tl Add e Define a new color in the same way as in the Configuration Control window see page Fig 4 239 Channel Colors window 4 51 03 06 B 45 0019 e 4 227 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 4 Select Zoom This function allows to change the zoom factor of an image displayed Click on Zoom will display the Zoom toolbar Any changes done with this toolbar are effective immediately The image can be
287. elow is the example of the acquisition of an image using an excitation wavelength of 532 nm and a fluorescence emission range above 550 nm Let the specimen be a thin section through a stem of Convallaria majalis Lily of the Valley The description applies to the use of the Axio Imager Z1 microscope and analogously also to the Axiovert 200 M 4 14 1 1 Requir ements T Configuration Control Channel Mode Lambe hiode DARE Ringernntng The suitable laser is switched on Close The specimen has been positioned and focused for Single Track Multi Track Ratio scanning Beam Path and Channel Assignment e The LSM button has to be activated either in RT Scanner Camera the LSM software or on the LSM button on the LSM tube itself l e Click on the Config button in the Acquire m subordinate toolbar of the Main menu BP 500 525 a This opens the Configuration Control wers Z f mR window e Click on the Single Track button _ A A cao e Click on the ChL1 icon and assign a color to Channel 1 in the Channel Color Selection window Activate channel 1 via check box Mirror Z e Click on the Pl icon of emission filter 1 before ChL1 and select the LP 550 filter Specimen e f required deactivate all the other channels ChL2 via check box Fig 4 305 Configuration Control window 03 06 B 45 0019 e 4 315 OPERATION LSM 5 LIVE Carl Zeiss Image Optimization LSM 5 LIVE DuoScan Excitation e Click on the
288. emove button An entry marked in ROI Lists stored ROI configuration is deleted Add to Lists The Add ROI List window is opened button 4 90 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 1 ROI Lists panel In the ROI Lists panel all the currently defined and stored ROI configurations are shown e Click on the ROI configuration which you want to use for the scan procedure The selected ROI configuration is highlighted in blue and displayed in the opened Image Display window e To produce a new ROI configuration an already stored configuration can be activated changed and stored under a new name using the Add to List button e To delete a stored ROI configuration from the list click on its name first highlighted in blue and then on the Remove button 2 Interactive ROI Definition panel In the Interactive ROI Definition panel the parameters of the ROI configuration just selected from the ROI Lists panel are displayed Furthermore it contains all the functions required for the creation of ROIs The X and Y values for Center Position and Dimension can be edited e Activate the relevant text box with a mouse click and enter the new value via the keyboard e f you click outside the edited text box the new value will be taken over and the ROI figure be shifted to the new position The upper part of the panel gives an overview of all the individual figures stored under the selected name according to typ
289. en ee E 4 287 AAs VIC AS UPSTATE UA CUO sese r a T 4 294 413243 3D Me s rement FUNGUONS sreaseioioise neri a E E E 4 304 41 249 EXPO Dala cn a a a a a 4 308 Al 2AT TOPO ROU O reee nE E EEE a ESAN EEEE TAE EERE 4 308 4 13 25 BIEN a EEE EE EE E E A E T T AA T E eee 4 309 4 13 25 1 Context Menu for Scan Information Text ccccccccccccceeeccceceeee ee ceeeeeeeeeeeeeeeesaneeeeeeaas 4 310 4 13 25 2 Context Menu for Topography IMages lt 2 6 cect Asie esheets as 4 310 4 13 25 3 Arranging and Printing the Print Preview asanseseanueneannussnrnreserrrssrrrissnrrrssnrrrrsnren 4 310 ANZ BPa SUI e A T Guach aes eanatcindacaten beanenee concent 4 311 4 13 27 Additional Display Mode in Time Series cccccccc cee cccee eee ceceee ee eeeeeeeeeeeceeeeeeeeeeaeeeeeeeaaes 4 312 ANS 27 ell DED V MEA dens hacts ibibo N heb imation in henmadmenceict obs manatenleue nancial 4 312 4 14 image Optimiza tION issi a a E a aa 4 315 4 14 1 WOECH nana e a A E ee 4 315 4 14 1 1 REGUIrEMEN IS miake a a a aaa e Bh eeana cays 4 315 4 14 1 2 Confocal Aperture Detector Gain Ampl Offset esneesiieerirerrrrreerrrrerrrrrerrrrn 4 319 4 14 1 3 Exposure Time Scan Average and Pixel Depth cssnesinessinsssiresriresrrresrrrerrrrerrrrerrn 4 321 4 14 2 STATE Wun 6 etc ay a a horna a a A E S 4 322 4 14 2 1 REGUIrEMEnN ESitianistqrenrens sate inaa aa ia a ck iaaa 4 322 4 14 2 2 Mage Opimizat Oeae n a e a a 4 323 4 15 Shut D wn Proced re saa a E a
290. ence of parameter changes in an Image Display window The Slice slider enables you to select the slice which shall be displayed in the Preview Image Display window e To start computation of the stereoscopic image click on the Apply button The image is built up twice once each for the red and green colors resulting in a Stereoscopic image 3 The stereoscopic effect can only be seen with the aid of red green 3D goggles The red lens is to be used for the right eye and the green lens for the left eye The presentation can be modified by selecting the Split Images Mode option in the Projection tab of the Stereo Images panel e By clicking on the Apply button the two stereo pairs are presented side by side and can be viewed without red green 3D goggles 4 162 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan 3D View Menu Carl Zeiss 4 7 4 3D DeConVolution DCV The 3D Deconvolution option is used for the resolution enhancement of fluorescence image stacks When a three dimensional object is reproduced by an optical system the resulting image of the object does not correspond exactly to the object s actual form The image of the object is distorted as it passes through the optical system In physical terms the actual object is convolved by the optical system s Point Spread Function PSF Real Object Imaged Object Specimen Mathematical Sign for Convolution Fig 4 161 Point Spread Function P
291. ength cm Width cm Height cm Weight kg Passively damped anti 145 115 115 150 vibration table System Electronic Rack and 135 100 300 Laser module Additional Hardware 135 61 100 Components 03 06 B 45 0019 e 2 9 LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE Carl Zeiss Microscopes LSM 5 LIVE DuoScan 2 11 Environmental Requirements 1 Operation specitied performance T 22 C 3 C without interruption 24 h a day independently whether system is operated or switched off 2 Operation reduced performance 10 C to 35 C any conditions different from 1 ie 5 3 Storage less than 16 h T 40 C to 55 C 4 Storage less than 6 h T 55 C to 70 C 5 Temperature gradient 0 5 C h 6 Warm up time 1 h for high precision and or long term measure ments gt 2 h 7 Relative humidity lt 65 at 30 C 8 Operation altitude max 2000 m 2 12 Vibrations Vibrations under operation conditions Shipping shock LSM 5 box with system table 5 um pp at 5 Hz 10 um pp at 10 Hz 10 um pp at 20 Hz 2 10 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 2 13 Microscopes Z motor Piezo Objective focus 03 06 LSM 5 LIVE SETUP REQUIREMENTS Microscopes Carl Zeiss Inverted Axiovert 200 M SP Upright Axioskop 2 FS MOT Upright Axio Imager Z1 All ICS objectives from Carl Zeiss and their accessories can be accommodated DC servomotor opto electronically coded Least Z interval 50 nm
292. ening of a database the middle record set is displayed 6 Show first record set at opening of database On opening of a database the last record set is displayed 7 Use separate path for Create and Open This option permits the path to be changed when the Open or New database function is used a Save most recently used path at exit and reuse at next program start On activation of this option the path setting last used is automatically selected again in the Open Database or Create New Database window b Use the following path at program start On activation of this option the path for the Open Database or Create New Database window can be entered directly in the relevant selection box or selected by clicking on the button in the Choose Directory window This path will then always be set when a database is opened or created Clicking on the User default path button firmly sets the C users default path 4 202 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 9 2 4 Database Table Viewer The Database Table Viewer tab permits the definition of the columns for the table display of a database This only requires the relevant check box to be activated with a click of the mouse On activation of the Automatic column width calculation option the column width is calculated automatically On activation of Save and use interactive column width setting the column width in the database can be matched as required Th
293. er filter feature is to be linked with the already defined one proceed as follows e Activate the relevant entries under Field and Operator and enter a value or text e g Name Convallaria under Value cs If groups of letters shall be searched the sign can be entered for undefined letters e g if you search for the letter row Conv enter Conv e Activate the required link type And Or or Not with a click of the mouse e g And e Click on the Add button The created filter feature is added to the work box of the Edit panel e g AND Name Convallaria The Modify button enables you to edit an already defined filter feature e Activate the required feature on the work box e Make the necessary changes under Field Operator and Value Select the link tyoe And Or or Not e Click on Modify The filter feature will be changed accordingly The Delete button enables you to delete a defined filter feature e Activate the required feature in the work box e Click on Delete The filter feature will be deleted from the work box e Clicking on OK will activate the filter the entire set of filter features displayed in the work box and close the Filter window On Filter is activated right on in the Database window and the filter function will be performed Only those images which fulfill with the defined filter features will then be displayed The procedure is interrupted via Cancel 03 06 B 45 0019 e 4 27 OPERATION LSM 5 LIVE Carl
294. erforms morphological operations on image sequences Erode Dilate Open Close Segmentates an image sequence to propose measurement Interactive Automatic Combines two image sequences by Boolean operations And Or Not Xor Mask B 45 0019 e 6 5 Carl Zeiss ye Scrap re Fill Holes View Menu Set Channel Colour Properties 2 Render Measurement Menu AI Automatic Object Windows Menu Arrange All Display Help Menu Content About 3D for LSM Tool Bar 3D FOR LSM User Interface LSM 5 LIVE LSM 5 LIVE DuoScan Selects or deletes objects of a defined size Fills holes in objects The colour and the weight of the single channels can be defined The properties of the image e g scaling use laser etc are displayed Calculates 3D reconstructions of an image sequence Surface Alpha Measures geometrical and densitometrical features General Object Features Volume Features Condition Arranges the windows automatically The current image is displayed in this window Opens the help for the software Displays status and release message of the software This bar provides buttons with iconized images of nearly all functions Clicking on one of the buttons will open a dialog window to define the function parameters Selecting an entry from the menu alternatively can activate the same functions Placing the cursor on a tool bar button will show a short description if the window
295. ers the PC system the power supply units cable connections safety interlock devices and other system components Please note that the LSM 5 LIVE are high precision opto electronic instruments Inexpert handling may easily impair their function or even damage them The openings for ventilation must not be covered There are hot surfaces on the HBO and HAL lamp When sliding the compact Laser module in and out of the System electronic rack take care not to catch your fingers The HBO 50 and 100 W XBO 75 W and X cite 120 lamps used on the light microscopes incident light path emit UV light which is harmful to the human eye and skin when observed without appropriate filters Never remove the lamp and look direct into the emitted light After installation or conversion of the LSM system authorized specialized staff must carefully check that it is in a proper condition and particularly that covers protecting against laser radiation are provided Tube openings or other unused mounts should always be protected against dust and moisture with the corresponding device components or with termination covers blind plugs By establishing a corresponding workplace environment please ensure that the formation of electrostatic charges of electronic components is avoided To avoid vibrations during operation the LSM 5 LIVE should only be operated in conjunction with the system table vibration damping 03 06 B 45 0019 e 1 13 NOTES ON DEVI
296. ess is needed to the long side of the rack Hence approx 50 cm space should be provided to the next wall Use of the LSM 5 LIVE is mainly done by operating the software Consequently we provide an ergo nomical computer workplace that fits the microscope table and should best be positioned on the right side of it We recommend the use of the 45 triangle extension to get the best ergonomical setup of the complete system but also a 0 or a 90 orientation to the microscope table is possible The best overall system design depends on the shape of your room see below 2 3 Room Requirements The LSM 5 LIVE needs approx an area of 7 m2 The room for the LSM 5 LIVE should therefore offer a minimum size of 10 m2 Due to the modular design both square shaped rooms approx 3 2 m x 3 m or rectangular shaped rooms approx 2 6 m x 3 9 m match the setup requirements of the LSM 5 LIVE Due to the modern diode technology of the lasers the system will produce much less heat and noise than older contocals Nonetheless an lab typical air condition or ventilation should be provided A constant temperature of 23 C would be ideal for constant and best system performance Details should be discussed with your local Zeiss LSM specialist 2 2 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE DuoScan Power Requirements Carl Zeiss 2 4 Minimum Space Requirements of the System For a list of the components dimensions see below in gene
297. eters 4 i a Time series 4 192 Loads a Beam Path Configuration form the Configuration list Use the pull down menu in the Property Value list to select the Configuration that should be used for imaging Changing between Single Track and Multi Track allows to chose between the different Configuration lists Multi Track also lists tracks that are used for Emission Fingerprinting or Online Fingerprinting Depending on the type of track different settings can be loaded optionally If they are not loaded and the parameters list of the Scan Parameter block does not offer them as being editable for example detector gain and offset default values will be used The list of properties lists all parameters that can be adjusted for the scan procedure The value for each parameter can be typed in individually Clicking the Read Back button will take over all current settings from the main software The check boxes next to the parameters can be marked individually or clicking Use all or Use none they can be all checked or unchecked with one click Only the parameters with a mark in the check box will be taken into account and adjusted when the Scan Parameter block is set in the program flow column This allows to change only some of the parameters for example the zoom at a certain position within the program flow as for each block the check boxes can be set This block performs the scanning of the sample according to the settings chosen with Be
298. eters to the default values A click on the Any View button displays the 3D reconstruction image in a shadow projection where the viewing point can be defined In addition to the zoom setting the image can be rotated around the three orthogonal axes via the relevant setting wheels 4 270 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss However the 3D orientation can also be set directly in the Image Display window by clicking holding and dragging the 3D reconstructed image with the mouse The following additional buttons are available in the Any View shadow projection mode e After activation of the Frame button below the image a bounding box is drawn around the 3D reconstructed image 3 Depending on the used mode and hardware configuration it can take several seconds until the 3D reconstruction is refreshed on the monitor after reorientation e A click on the Coordinate System button displays a colored coordinate system in the Image Display window where the X axis is displayed in red color the Y axis in blue and the Z axis in green e Aclick on the Scale button display an X Y and Z scale in the Image Display window e Aclick on the Home button resets the display parameters to the default values e A click on the Animation button activates the animation mode The object can be pushed by dragging in the Image Display window and rotates continuously Any new push with pressed l
299. eview The Preview function enables you to preview the result of the defined Add operation in a preview window Fig 4 111 Preview panel e Activate the Preview check box with a click of the mouse The Add Preview Image Display window is displayed with the operation result e Deactivate the Preview check box to close the Add Preview Image Display window fs After a change of the formula in the Add panel click in the Add Preview Image Display window for an update 03 06 B 45 0019 e 4 123 OPERATION Process Menu Carl Zeiss TS Subtract Close Click into window Appl Channel Chi aa Click into window Channel Ch Destination Click into window Mew 8 Bit New 12 Bit Channel Hep ka New Image 12 bit Source 13 00 Source 17 00 t 2 00 2 00 Fig 4 112 Subtract window LSM 5 LIVE LSM 5 LIVE DuoScan 4 6 2 Subtract The Subtract function links two channels each of one or two images into a new channel by Subtraction The channel created in this way can be stored via the Save As function 4 6 2 1 Open Close the Subtract Window e Click on the Subtract button in the Process subordinate toolbar of the Main menu This opens the Subtract window e Click on the Close button to quit the Subtract window 4 6 2 2 Performance of the Subtract Function This function is performed in the same way as the Add function see Add page 4 121 The onl
300. f the Z Stack not available in the Nearest Neighbour method If Auto detect is enabled the Restoration Effect slider is disabled The Iterative method permits in addition to the parameters of the Inverse method the maximum number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop function to be activated deactivated The Auto Stop function interrupts the calculation depending on the set image improvement delta between last but one and last cycle in no matter whether the value under Maximum Iterations has been achieved or not The Nearest Neighbour method permits entry of the Number of Neighbours and the Sharpness in Focus value in addition to the Restoration Effect Method PSF 2 PSF tab Numerical Aperture Objective 1 00 In the 3D Deconvolution option a theoretical point Magnification Objective 40 spread function PSF is calculated from the Refractive Index of Immersion Fluid 1 000 systems settings objective data wavelengths aperture size Channel ie The PSF data are displayed in the PSF tab In the am case of wavelengths above 700 nm the NLO Excitation 633 nm button is automatically enabled Emission E633 nm o n The displayed values are always taken over by the system data but can be edited subsequently for simulation purposes Fig 4 167 PSF tab 4 166 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss 4 8 Macro Menu The
301. f the trigger keys 1 to 4 e g Trigger1 In this example the scan procedure Is triggered on pressing key 1 of the trigger control and an out signal is given at the same time 3 When starting a Time Series via Trigger the Start T or Start B button must be pressed first Waiting for Trigger will then be displayed in the status line Then the relevant trigger key on the Trigger Control must be pressed to start the first scan procedure of the Time Series 2 Start via Time Manual Trigger Time For the start via the time set on the PC Time l l button activated the start time must be entered T 11 mm 00 ss 00 T r as 7 ne ii O first in the Time input box Fig 4 81 Start Series panel e Click in the Time input box to open it e Enter a start time via the keyboard Then click outside the input box once to close it again LB When starting a Time Series via the time the Start T or Start B button must also be pressed in this case Waiting for Start Time will be displayed in the status line The Time Series is started when the starting time has been reached The starting time for the Time Series can be changed online 3 Pre Scan The Pre Scan allows to image the sample continuously before actually starting the image acquisition Go fF This function should only be used for starting the time series manually After clicking the Start or Start B button the button Go appears on the lower right hand side of the control wi
302. ff filters for smoothing separation of roughness or waviness Geometry buttons Inversion various fit procedures hole filling Display buttons 2D Intensity Height Gradient Iso Lines z Map Intensity Black 3D Profiles Grid Filled Shaded Measure buttons Diagrams Profile z Distribution Bearing area ratio plot Slope distribution Roughness parameters Volume parameters 4 13 24 1 Channel Selection e Select the channel to be viewed using the relevant button e g Ch1 4 13 24 2 Generate Topography The three buttons provided in the Generate button bar allow you to generate the topography in different ways Maximum e Click on the Maximum button to calculate the topography surface by finding the maximum intensity value If the optical section with the highest intensity value is found the intensity values of both neighboring slices are also taken into account so that a 3 point maximum fit is calculated e Incase it happens that the maximum possible intensity value is present in more than one optical slice for a given pixel saturation the mid section of all saturated intensity slices is chosen as a reasonable approach Center e Click on the Center button to calculate the topography surface by using the center of gravity of all summed up intensities of the stack for a given xy print This mode provides better result for smooth surfaces of low intensity or nearly transparent surfaces The receiver gain
303. ffset Offset Offset 0 00 um Offset 0 00 um Fig 4 72 Scan Control window Mode settings for camera control 2 Function description Mode button Displays the selected objective frame size and pixel depth Frame Size Selects between square formats or free defined frame sizes Format Selects between a range of default camera resolutions The 5x5 binning mode can be used for focusing without delay of the image display Data Depth Sets the pixel depth Zoom Offset Shifts a subregion in the frame Reset Resets the frame subregion to default value selected in Format 4 88 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss A Scan Control 2 x 2 settings ti mj rF ETE E Frame Hs ae sie Exposure Time 4 ee eee gt j 0 01 sec Fig 4 73 Scan Control window Channels settings for camera control Channels button Displays the activated channels and possible settings Exposure time Find Fast X Y Single Cont Crop Info button Close 03 06 Sets the exposure time of the camera Starts a prescan and sets the exposure time automatically In case of a camera multitracking only one channel should be selected in Configuration Control in order to speed up the find function Starts a fast online scan mode e g for focusing Also the 5x5 binning mode can be used to be set in Mode Format Starts a single image acquisit
304. fset The offset of the scan area from the center of the field of view is displayed online in um for X and Y A click on the center button will recenter the scan area to the field of view Clicking holding and drawing the rectangle with the mouse permits the scan area to be moved directly within the field of view Recommended setting to start with Offset X 0 Y 0 LS During the scan procedure the functions Objective change Speed Scan Corr Zoom Rotation and Offset can be influenced online By clicking on the Reset button the scan zoom is set to 1 and the XY offsets are set to the zero position 4 74 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 2 Channels 1H Scan Control KE If the Channels button is activated the Channel Settings and Excitation of Track panels are displayed in the Scan Control window ee Channel Settings Channels chet ieee Channel Settings panel In the Channel Settings panel the channels incl ratio channels defined in the Configuration Control window are listed track by track as KA selectable buttons Bis EE gt 4 Max e Optical slice lt 14 9 pm Pinhole 0 1 03 Ain Equival Depending on the selected Channels button e g rr ChL1 T1 the currently used settings of Pinhole ooo EA gt confocal aperture Detector Gain Amplifier Offset Eipoaute Tine 7 4 z F and Amplifier Gain are displayed
305. g 4 217 Save tab Image files to subdirectory of the database Database and image files to the same directory At Create Database automatically create a subdirectory with the same name as the specified database and create the database and image Tiles in that subdirectory If the Save prompt at closing modified windows check box is activated you are automatically asked on closing a changed Image Display window whether the image shall be stored If the Warning before overwrite existing recordsets check box is activated this question is asked automatically on storing an image under a new name If an image file with this name already exists in the database If the Remember Name Description and Notes in the save dialog check box is activated the Name Description and Notes text boxes of the Save Image and Parameter As window show the text for the image last saved You can edit the text boxes as required for the new image to be saved If the Remember Name Description and Notes in the save dialog check box is deactivated the three text boxes are blank when the Save Image and Parameter As window is opened 4 208 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Options Menu Carl Zeiss 03 06 B 45 0019 e 4 209 OPERATION LSM 5 LIVE Carl Zeiss Maintain Menu LSM 5 LIVE DuoScan 4 10 Maintain Menu The Maintain menu contains additional modules to check and guarantee the interference tree operation of all the sof
306. g shapes numbered 1 to 3 from left to right are available amp If Grey Morphology is selected the function will respect all grey value shades of the sequence Input If Grey Morphology is not selected the function will distinguish between O and non O only The result Output will be a binary sequence Parameters Input Input image sequence Output Resulting image sequence Shape Shape used 1 Cross 2 cube 3 cube cross Count Number of recursive operations Grey Morphology 0 Distinguish between 0 and non O only 1 All grey value shades are taken into account 03 06 B 45 0019 e 6 29 Carl Zeiss 3D FOR LSM LSM 5 LIVE Functions LSM 5 LIVE DuoScan Morphology Dilate This function dilates structures in an image sequence HH Horphology Erode Dilate Open Close Input i All 1 Output 2 New Shape Ba Ee Count Poo w ppt 0K Cancel IY Grey Morphology Fig 6 21 In the Morphology dialog window the tab sheet Dilate must be selected Dilation makes bright regions larger on a dark background It also results in the filling of gaps and smoothing of small contour details If Input is a multichannel sequence any number and combination of channels can be selected Output will only get the selected channels as results The Input sequential image is dilated Count times with the shape Shape The Count scroll bar determines the number of recursive operations The follo
307. ght mouse button closes the figure and ends the procedure EN Open free shape curve button Creation of an open Bezier figure in the Image Display window The first click sets the starting point each additional click adds a further line a click with the right mouse button closes the figure and ends the procedure 03 06 B 45 0019 e 4 231 Carl Zeiss Ol 4 232 OPERATION LSM 5 LIVE Display and Analysis of Images LSM 5 LIVE DuoScan Circle button Creation of a circle in the Image Display window Clicking and holding down the mouse button sets the center point drag the diameter and release the mouse button to end the procedure Line with arrow button Creation of a line with arrow in the Image Display window Click and hold down the mouse button drag the line in any required direction release the mouse button to end the procedure Scale button Creation of a horizontal or vertical scale with default increments in the Image Display window Click and hold the mouse button for the starting point drag horizontal or vertical scale release the mouse button to end the procedure Gray tones color shades button Generates a rectangle with a display of gray tones or color shades in the image Color shades are displayed if a palette has been loaded with different colors being assigned to the gray tones A Text button Creation of a text box in the Image Display window After clicking on A the Text window will be displayed
308. guide pins 7 1 5 and secure it with the three screws 7 1 1 ms As the Scanning Module is heavy weighing about 19 5 kg it is easier if the changeover is carried out by two persons e Pull off covering caps 7 1 3 from the CAN BUS and RS232 interface ports at the rear of the Axiovert 200 M remove the two cables 457411 9011 CAN BUS and 457411 9012 RS232 from the Axioskop 2 FS MoT plug them into the Axiovert 200 M and secure them there e Switch the LSM 5 LIVE on e Click on the Stand select icon to update the system database with the new database of the Axiovert 200 M microscope e Restart the LSM 5 LIVE program 03 06 B 45 0019 e 7 5 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX Detaching Attaching the Scanning Module Carl Zeiss z ap lt U N Q T O Dm Z Z U VY N Q U gt oO gt O U M Change over of the Scanning Module Fig 7 1 03 06 B 45 0019 e 7 6 LSM 5 LIVE ANNEX LSM 5 LIVE DuoScan Hints on the Use of the Piezo Fine Focusing Stage Carl Zeiss 7 5 Hints on the Use of the Piezo Fine Focusing Stage 7 5 1 General Description The Piezo fine focusing stage is a compact attachment for the Axio Imager Z1 and Axiovert 200 M microscope stages which allows the particularly fast and high precision fine focusing of the object The Piezo stage permits fine focusing over a range of 250 um with the smallest step width being less than 10 nm reproducibility be
309. h one of the recommended objectives should be part of the shipment in order to be able to repeat the Basic Calibration without calibrating all objectives again see objective list down below ls for calibrating both collimators RGB V Is for calibrating the Y position of the Beam Shift Compensator BSC for all present NFT positions Ge On currently delivered LSM 5 LIVE Systems the Basic Calibration is done in the factory This can be confirmed by checking that BasicCalibrationLog txt Is present in C AIM Bin Objective list objectives are listed in order of most to least recommended Plan Apochromat 20x 0 75 Plan Neofluar 40x 1 3 Oil C Apochromat 10x 0 45W C Apochromat 40x 1 2W corr Plan Apochromat 5x 0 16 Plan Neofluar 10x 0 3 Plan Apochromat 63x 1 4 Oil C Apochromat 63x 1 2W corr Plan Neofluar 20x 0 5 03 06 B 45 0019 e 4 177 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan B Calibration Matrix Is based on the calibration results of the Basic Calibration Needs to be performed for each objective separately Is for calibrating the collimator position of both collimators RGB V only for the selected Objective depending on Laser Line Zoom EF Filter Position ls for calibrating the BSC Y position depending on Laser Line Zoom EF Filter Position re On currently delivered LSM 5 LIVE Systems the Calibration Matrix
310. he Base directory selection box Furthermore a name for the subdirectory must be entered in the Subdirectory base name input box The starting value for the images then created to which a continuous number is automatically assigned is set in the Subdirectory counter input box 03 06 B 45 0019 e 4 201 OPERATION LSM 5 LIVE Carl Zeiss Options Menu LSM 5 LIVE DuoScan Autosave ee D jatabase General i Database Table Viewer Database Gallery Viewer 4 9 2 3 D at a b ase G enera 2 Start with Form 2 Show first recordset at opening of database Start with List Show middle recordset at opening of database C Stat wth Goley F Show last recordset at opening of database This tab permits the basic starting settings for the MV Use separate path for Create and Open use of d ata b ases Save most recently used path at exit and reuse at next program start Use the following path at program start C Lsm61 Obilder convallaria manual mdb m User default path 1 Start with Form On opening of a database the Form option is displayed first Fig 4 203 Database General tab 2 Start with List On opening of a database the List option is displayed first 3 Start with Gallery On opening of a database the Gallery option is displayed first 4 Show first record set at opening of database On opening of a database the first record set is displayed 5 Show first record set at opening of database On op
311. he Basic Calibration takes about 15 min z e The displayed curve should resemble a Gaussian profile E aai e After the macro has finished a message will mm pop up with the request to restart the LSM a 7 i OO software to make the changes effective Fig 4 179 Collimator Calibration Window e Press Exit and restart the LSM software 03 06 B 45 0019 e 4 181 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan Running the Calibration Matrix i If you haven t selected the check box for Basic Calibration the Calibration Matrix will be selected automatically In this case when the Calibration Matrix is running the Basic Calibration is disabled e This window provides three check boxes Fig 4 180 e The recommended setting is to select Create new calibration file All previous files will be deleted Exceptions No check box checked Especially after an unexpected interruption of the program it is possible to continue from the position where the process stopped i saving time S E Overwrite Collimator values a i Seon serar rearen uenra enne ine arran The values for the chosen objective will be added or updated Tanho oan bar ia e E After selecting the Collimator option the Fig 4 180 Running the Calibration Matrix Pinhole Y option will be selected automatically e Double check if the objective settings shown at the b
312. he following function buttons are available in the lower row of the Zeiss LSM Image Examiner main menu Projection button One single projection or a series of projections can be calculated after rotation of the data package about the X Y or Z axis DepthCod button The depth information contained in a sequence can be colored with the colors of the rainbow Stereo button Stereoscopic images can be generated Ratio button Permits two channels to be linked into a new channel by the creation of a ratio Copy button Permits one channel each of an existing image to be copied and stored as a new image Filter button Permits the subsequent processing of scanned images via the integrated filters Interpolate button Permits the continuous contrast and brightness change in a stack or time series through interpolation between the starting and end values Shift button Produces a congruent image with relation to the pixels of the various channels Duplicate button Permits images including Z Stacks and Time Series to be duplicated completely Contrast button Permits the subsequent modification of contrast and brightness of the stored image These functions correspond to those of the Expert Mode of the LSM 5 LIVE software and have already been described in chapter 4 5 0 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Contents Carl Zeiss CHAPTER 6 3D FOR LSM CONTENTS Page 6 SD TON SI eca swacaceteasenacens wecaudacocmeee A AEN
313. he pixels of the created line to which the relevant gray value is assigned now appear in the Profile window At the same time the distance of the created line is displayed in um for checking Any required number of lines can be defined in the image The previous line is deleted e A click on Cancel will end the macro 03 06 B 45 0019 e 4 175 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan hitiem neiaa z ae 4 8 4 3 Multiple Time Series Macro The Multi Time Series program is designed to provide flexible programming of automated time dependent experiments Apre program Cycle Delay Apre bleaching functionality not available Leng ere rar An autofocus functionality A number of different single time series vea p HPO l 7 PBlock 1 Parami f j 4 called a block either at the same position of Autotocus motorized stage is available M Aai per wee cer per rt mn zow fF rof om A time series of a tile scan Tieg d b E Sheree a a Pein l Bleach ftv Contig Fev o M Spoe mir Tiree Dely ta fioi al j f ma PCH peor view es Configuration Time Interval and Number of Scans Singe Tasch Gr Hidi Track panara Birn A time series of a tiled Z stack gt F wava a He E a sample or at different positions if a Vv Flplinsh A Z Stack with triggered acquisition of each single image in the stack The basic programming
314. he suitable lasers are on Beam Path and Channel Assignment spectra e The specimen has been positioned and focused ts uscan pe for scanning e The LSM button has to be activated either in the LSM software or on the LSM button on the LSM tube itself Contig A Erts In the following example 2 Channels shall be activated for the scanning procedure one for ress 7 488 nm using emission filter BP 500 525 and one for 532 nm with LP 550 NFT 532 is used as the secondary dichroic beam splitter P 415 480 m l LF 505 TER f Chi l B a ChL2 NFT x Z VA Specimen e In the Acquire subordinate toolbar click on the Config button Q This opens the Configuration Control of o0 window Fig 4 316 Configuration Control window e Click on the Single Track button e Activate in the same way as for the single channel see page 4 315 channel 2 ChL2 the indicated emission filters and the main and secondary dichroic beam splitter for the scanning procedure The contiguration loaded is displayed in the Beam Path and Channel Assignment panel e Click on the Scan button in the Acquire subordinate toolbar of the Main menu This opens the Scan Control window e In the Scan Control window set the parameters in the same way as described for single channel presentation e Click on the Find button in the Scan Control window This starts the scanning process The scanned image appears in a separate wi
315. hen the end time has been reached If the entered Number of cycles has been processed the Time Series is finished If the number of cycles has not yet been processed the Time Series is only interrupted Waiting for Start Time is displayed in the status line The Time Series can now be continued by entering a new start time or finished via Stop The end time for the Time Series can be changed online 4 5 6 4 Cycle Delay Time Interval Panels Appl 5 D l l a EE Depending on the settings in the Time Series tab see Options menu Settings the time series interval is defined either as a Cycle Delay or Time Interval Accordingly either the Cycle Delay d l n i k panel or the Time Interval panel is displayed in sii MUS the Time Series Control window Trigger in None Trigger out None l Cycle Delay is the interval between the end of one scan process and the beginning of the next Fig 4 85 Cycle Delay panel Time Interval is the interval between the meinen beginning of one scan process and the beginning of the next Apply Store Delete The Cycle Delay or Time Interval panel permits 00 msec cl ella the intervals to be activated and changed 0 0 msec 0 0 msec 0 0 msec me PU Unit min sec mz 0 0 msec Trigger in None Trigger out None Fig 4 86 Time Interval panel 4 98 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss
316. hk ita varadi subordinate toolbar of the Main menu Hias Image 1 2b ees Channel her This opens the Ratio window e Click on the Close button to quit the Ratio window oc m D 37 60 10 7 Ga 0 4 6 4 2 Performance of the Ratio Function This function is performed in the same way as the Inkarwihy tanga 0 4 Opecsbon Lit Operator Add function see Add page 4 121 However self edited formulas can be used for F Biever image calculation Fig 4 114 Image Calculation window 4 126 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 6 4 3 Formula Panel The formulas can be typed in using the keyboard Click the Operators button to open the permissible operators list Select the operator s trom this list Fig 4 115 and click the Insert button The operators will be inserted into the formula at the cursor position By highlighting the selected operator a description of the function of this operator is displayed in the lower part of the operators list The calculation works pixel by pixel and starts with the upper left pixel irregardless of the image size of the two images or image series Ike 4 6 5 When choosing images of different data depth the check box next to Intensity range 1 0 should be marked The image intensity for all images will then be normalized to values between 1 and O Copy Channel The Copy function permits one channel each of an exi
317. iagram above is a schematic representation of the LSM 5 LIVE system Laser light is focused onto the specimen through an objective in a diffraction limited mode Light emitted at the focal plane and at planes below and above it is directed via an Y scanner onto the Achrogate beam splitter which separates the emissions from the excitation light The fluorescences are separated from each other by a Secondary Dichroic Beam Splitter and directed to individual photodetectors CCD Line1 and CCD Line2 3 4 B 45 0019 e 03 06 LSM 5 LIVE INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 LIVE DuoScan Performance Features of the LSM 5 LIVE Carl Zeiss 3 4 Performance Features of the LSM 5 LIVE 3 4 1 Optical and Mechanical Aspects The highly integrated system design makes for the shortest possible optical paths top grade optical precision and high stability The compact scanning module can be fitted to an inverted Axiovert 200 M SP or upright Axioskop 2 FS MoT Axio Imager Z1 Axio Imager M1 microscope in less than three minutes On the Axiovert 200 M the scanning module may be mounted either to the base port directly below the microscope or to the side port For the VIS visible light Laser Module the user can select from up to four lasers with wavelengths of 635 532 488 440 and 405 nm Coupling of the laser light is through polarization preserving single mode optical fibers One beam collimator for the visible ranges provides optimum adaptatio
318. iagrams MV Scan the whole image geeeeesenesesesesessccssssssssssssscesesssesssesesesesssssseesey C ROls diagrams I Save the whole time series Necccsssscsesesessssnssesseeseseessnscnensnenanssasassansnaeses Fig 4 212 Scan Mean of ROIs tab Fixed time range for diagram time scale input of the time range in seconds via input box Fixed number of cycles for diagram time scale input of the time range in number of cycles via input box 03 06 B 45 0019 e 4 205 OPERATION LSM 5 LIVE Carl Zeiss Options Menu LSM 5 LIVE DuoScan 2 Initial diagram types The following settings are possible by activating the relevant option button One diagram Channels diagram ROls diagram On activation of the Black graphs b check box the intensity profiles in the diagram are displayed in black monochrome 3 Live image a Scan the whole image If you activate this check box the complete live image will be scanned only the defined ROIs will be scanned if the check box is deactivated b Save the whole time series If you activate this check box the complete Time Series will be scanned only the Mean of ROI series will be scanned if the check box is deactivated poeeesssessssssssessseceseceseesessneessssssesessscssssssssesenes Recording Reue Tmeseies Tempoiay Fies Scan Mean of Rls 4 9 2 13 Temporary Files m Image Memory i C Use RAM i Use Disk temporary fie The Temporary Files t
319. ic Rack and the power supply switch of the Ar Laser to position OFF after 5 minutes This puts your LSM 510 microscope system including the computer off power 4 15 4 Turning off the HBO 100 e Switch off the HBO 103 via the toggle switch of the HBO 100 power supply 4 326 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Software and Hardware Options Carl Zeiss 4 16 Software and Hardware Options This section describes optional software and hardware configurations Depending on your configuration the content of dialogue and function may differ 4 16 1 Software The following software packages for Release 4 0 are available Software Physiology Software Topography Software Macro Recorder and Editor Software 3D for LSM Software Multiple Time Series Software Image VisArt Software Deconvolution Software StitchArt plus Software Visual Macro Editor Software FRAP If your configuration does not include any of the SW packages Physiology FRAP or FRET the following functions are not available Mean of ROI button in the Image Display window If your configuration does not include the Physiology software package the following functions are not available Mean of ROI scan button in Time Series Control lon Concentration button in the Process Menu If your configuration does not include the Visual Macro Editor software package
320. ic collimator adjustment Available for most common objectives Collimator Collimator LIYE M Description Collimator LIVE A Position mm 4 55 Ado H Store Current Dy Move To Stored E a TA Move ia Fig 4 229 Collimator panel B 45 0019 e 4 217 OPERATION LSM 5 LIVE Carl Zeiss Maintain Menu LSM 5 LIVE DuoScan 4 10 3 4 Aperture and collimator Adjustment Adjustment of the LSM 5 L VE apertures can be performed manually or automatically If several channels are used to produce the image all the used apertures must be adjusted separately 1 Manual aperture and collimator adjustment The position of the aperture relative to the detector in terms of Y coordinate contributes substantially to image optimization Requirements to make aperture position changes visible immediately The image must be scanned by the continuous scan method Select a fast scanning speed Measurement with Average Number 1 only no averaging of several measurements On the Channel Settings panel click on Channels button in the Scan Control window select the aperture size so as to have the best possible image contrast e Click on the Pinhole button in the Maintain subordinate toolbar Pinhole LIVE_PH2 Seenition E he e Select the aperture pinhole to be adjusted from the Description list box Diameter um 71 4 H e Use the Diameter slider to set the smallest Pi
321. ic tab sheet of the Contrast dialog window must be selected This function enhances the contrast of an image sequence by spreading the grey value distribution over the maximum possible range This function is used to achieve a better view of an image The light and dark grey value ranges with a low share of pixels are excluded from the operation by the parameter Threshold The Threshold units are in thousandths of the total number of voxels Using a value of 10 means that the scale interval is set so that 5 1000 of the total number of voxels on the light side and 5 1000 of the total number of voxels on the dark side of the grey value distribution are excluded Input indicates the sequence to enhance If it is a multichannel sequence a single channel all channels or any number can be selected The Input histogram shows the grey value distribution of the selected channels of the Input image sequence Output defines the name of the result sequence It will get only those channels which are chosen by the Input parameter The buttons labeled with 8 and 12 define the grey value intensity resolution in bit Normally the result will get the same resolution as the Input sequence A change will be needed if image sequences with different resolutions should be combined Rising the grey value range to 12 bit will not enhance the display quality or measurement accuracy The smooth and morphology functions will produce results with finer gradations 03 06 B
322. ick on the z Histo button in the additional button bar now displayed The lower part of the Image Display box shows the 3D height distribution of the topography Abija A 1000 TE Binet D a5 10 15 ilp oo Resi VOM a 10a J6 1 charred 12 ba Fig 4 297 Image Display window Topography display 2D Histo 3 Curve of tp measurement mode in 2D display e Click on the Diagram button in the Measure button bar Click on the Curve of tp button in the additional button bar now displayed The curve of the bearing area ratio as a function of the height is displayed below the image also see 3D measurement functions page 4 304 4 298 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 Grad Histo measurement mode in 2D display e Click on the Diagram button in the Measure button bar Click on the Grad Histo button in the additional button bar now displayed The lower part of the Image Display box shows the gradient distribution of the topography Before creation of the slope diagram the image should be filtered at least once using a low pass filter since otherwise the rough height gradation of the image will result in a comb shaped histogram The Root Mean Square Slope RMS Slope parameter is calculated and displayed below the chart The following formula is used for calculation 2 2 l 5 x y 2x19 day 23 N 1 N 6 Ax Ay j l 5 R
323. icroscope must be in the LSM mode i e the LSM button on the tube of the microscope stand must be pushed or the LSM button in the Acquire Subordinate toolbar must be activated to set the LSM mode The scanning actions are started via the buttons on the right hand side of the Scan Control window and the scan parameters are set in the main part of the window An acquired image is displayed in a separate Image Display window If an Image Display window is not yet available a new Image Display window is automatically opened during the acquisition The following scanning modes can be performed Line scanning of a line in the XY plane Line Line Time Series scanning of a line with different Z values Line Z Stack Line Z Stack Time Series scanning of an XY frame Frame Frame Time Series scanning of XY frames with different Z values Frame Z Stack Frame Z Stack Time Series 4 68 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 4 1 OPERATION Acquire Menu Carl Zeiss Open Close the Scan Control Window e Click on the Scan button in the Acquire subordinate toolbar of the Main menu This opens the Scan Control window which shows all lasers connected to the system e Click on the Close button to quit the Scan Control window The following main function buttons are available in the Scan Control window Generally available buttons Mode button Channels button
324. ies list box Use the Drives list box to choose a different drive In case of choosing the TIFF format in the Files of Type box three number characters are always expected before the dot in the filename extension The first number must be 000 at the end of the filename From a complete sequence only this file is listed in the dialog if LSM TIF Images 000 tif Is selected in the Files of Type box To view all TIFF files All TIF Images tif in the Files of Type box must be selected This selection enables to start with a different file than with the very first named 000 tiT at the end of the filenames three number digits Currently the Carl Zeiss Vision file format KE Images 0 img is supported Two Tiles per channel are saved 03 06 B 45 0019 e 6 11 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan Carl Zeiss Vision image sequences must have a number digit at the end of the base filename They are used to indicate the different channels in a multichannel sequence The numbering starts with zero 0 If a sequence is saved in the Carl Zeiss Vision format the numbers are generated automatically To load such an image sequence KE Images 0 img in the Files of Type box must be selected The window incorporates the usual file selection controls The bottom half displays a selection of the image properties that are stored in the image sequence Parameters BaseName Base name of the TIFF Tiles image sequence to
325. iezo objective focusing device allows stacks to be produced considerably quicker than via the focus of the microscope stand The focus position remains unchanged e Clicking on the Leveling button moves the piezo objective focus to the zero position while the motor focus moves into the opposite direction at the same time i e the position of the object in relation to the objective remains unchanged This function is used to set defined initial conditions e Use the slider or the arrow keys to set the number of slices for the Z Stack e Use the slider or the arrow keys to set the size of the interval Num Slices and Interval can be varied independently of each other within the piezo objective focus work range of 250 um When change is made to Z Sectioning or vice versa values are also taken over provided they are within the piezo objective focus work range If a larger range is set for the Z Stack under Z Sectioning or Mark First Last the Interval is matched accordingly when changing to Hyperfine Z Sectioning while Num Slice remains constant e Use XYcont Line Sel and Range to determine the parameters of the Z Stack identical to Z Sectioning If the green line Current Slice is shifted after the creation of Range the focus position will change the piezo objective focusing device remains in the center position The red lines stack limits can only be changed symmetrically to the Current Slice position within the piezo objective fo
326. ight corner of each window This closes the respective window and removes the respective icons from the taskbar After all dialog windows have been closed the LSM 5 LIVE Switchboard window appears E Switchboard Fig 4 319 LSM 5 LIVE Switchboard menu e Click on the Exit button This terminates the LSM program The monitor screen shows the desktop of the WINDOWS XP operating system 03 06 B 45 0019 e 4 325 OPERATION LSM 5 LIVE Carl Zeiss Shut Down Procedure LSM 5 LIVE DuoScan 4 15 2 Shut Down the WINDOWS Operating System e Move the cursor to the bottom margin of the screen This opens the taskbar containing the Start button e Click on the Start button of the taskbar This opens a pop up menu e Click on the Shut Down item This opens the Shut Down Windows window in which you can select between Shut down Restart and Login e Unless already set by default click on Shut down the computer e Click on the Yes button About 20 seconds after WINDOWS XP has been run down and your computer turns off 4 15 3 Turning Power Off LS Please bear in mind that a cooling phase of at least 5 minutes is required between switching off of the laser via the software and switching off of the entire system via the REMOTE CONTROL main switch or the Power Supply switch of the Enterprise UV laser Set the REMOTE CONTROL switches System PC and Components or the Main switch on AN the System Electron
327. ilter List function permits updating of Summary DIC the filter data in the software after a change of filters on the stand e Close the LSM 5 L VE software program e Double click on the Change Filters icon on the desktop e Click on the Edit Filter List button in the Emission Filter amp Beam Splitter Control window The Edit Filter Beam Splitter List window is opened Fig 5 1 Edit Filter Beam Splitter List window This window permits a list of the most frequently used filter sets to be compiled e Click on the arrow button in the Filtername list box to open it e Select the filter set which shall be included in the list 5 2 B 45 0019 e 03 06 LSM 5 LIVE TOOLS LSM 5 LIVE DuoScan Change Filters Carl Zeiss e Click on the Apply button The selected filter set is included and displayed in the list below the Sumary list box This filter set is now also available in the Name selection boxes of the Filter Cubes Stand panel and can be assigned to a filter wheel position To remove a filter set which is no longer needed from the list proceed as follows e Click on the name of the filter set concerned in the list box of the Edit Filter Beam Splitter List window e Click on the Remove button The filter set is deleted from the list and is then no longer available in the Filter Cubes Stand panel of the Emission Filter amp Beam Splitter Control window Add New iwi Add New Filter Beam Splitte
328. image information is generated by adding up all scans pixel by pixel and then calculating the mean value 03 06 11 In the Sum method the pixel values of all scans are only added up without a mean value being calculated e Select the Number for averaging Continuous averaging is possible in the Frame mode In this case a Finish button for ending continuous averaging Is displayed instead of the Cont button 74 Scan Control E x Channel Settings Channels Chui Ch ot Pinhole Pa ar H DI 1 Max mA Optical slice lt 14 9 um Pinhole 1 03 Airy Equival Detector Gain o gt Amplifier Offset o Ado gt 5 a Time Ez al es gt 0 5 Frames per Second Fig 16 Scan Control window Channel settings e ST S12 chame tet Paper mag cite Dupay Zoon 1 Pasie Ho Palette Fig 17 Image window Adjusting the pinhole e Select Channels in the Scan Control window e Set the Pinhole size to 1 Airy unit for best compromise between depth discrimination and efficiency Pinhole adjustment changes the Optical Slice When collecting multi channel images adjust the pinholes so that each channel has the same Optical Size This is important for colocalization studies Image acquisition Once you have set up your parameter as defined in the above section you can acquire a frame image of your specimen e Use one of the Find Fast XY Single or Cont buttons to start the sc
329. imum roughness depth R Peak to Valley PV S z max Ania maximum height difference of the overall topography along a profile 4 300 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Classification of topography in 5 equal area elements rectangles in the 2D mode average roughness depth R R _ lt max E Zmin1 T lt max 2 E lt min2 Lmax 3 7 lt min3 Z max 4 7 Zmin4 a lt max 5 E Zmin5 e 5 Averaging of R values of all the 5 single area elements When combined both parameters provide information about the homogeneity of the surface Big differences are indicative of pronounced inclination of the overall area or of spikes Developed Surface Area Ratio X surface area X projected area The percentage of the 3D surface area sum off all triangles formed by adjacent data points to the 2D surface area produced by projecting the 3D surface onto the threshold plane maximum roughness depth Rmax n Riis Max Z g Z mini Kmax 2 E Lmin Lak a Zmin3 Amari g Zmin4 Z m x3 B Ze maximum of R values of all the 25 single area elements re Both the roughness parameters and the z histogram can be changed by using filters 6 Roughness measurement mode in 3D display e Click on the Roughn button in the Measure button bar The roughness parameters are calculated and displayed on the left below the image All roughness parameters calculated trom a 3
330. in the Channel Settings panel to optimize Channel 1 Optimization is performed in the same way as for the single channel and can be monitored online in the relevant separate image of the channel e Then optimize the second channel by clicking on the relevant button ChL2 in the Channel Settings panel of the Scan Control window Scan image 1 Scan image 2 Cumulative scan image Fig 4 318 Scan Control and Image Display windows Now effect image optimization as explained for the single channel mode but separately for each channel e Now click on the xy button of the Display toolbar The composite scan image of two channels is presented in a common window ci Image optimization can be effected much faster if you select a smaller frame since less data have to be processed 4 324 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Shut Down Procedure Carl Zeiss 4 15 Shut Down Procedure Never shut down the computer by its main switch while your LSM program is still active or else you will lose the currently set operating parameters and the images just scanned 3 In the Settings for user dialog window which can be activated with the Options Settings buttons activate Laser off or Exit in the Shutdown tab The lasers will then automatically be switched off when you exit the LSM program 4 15 1 Exiting the LSM Program e Close all open windows of the LSM program by clicking on the closing icon in the top r
331. in the Cycle Delay list box or Time Interval list box and click on Store to store the set of delays Activating delay time or time interval If required a set of delays or time intervals can be activated again quickly e Open the list box with a click on the arrow button and select the required set with a click of the mouse e Then click on the Apply button to activate the set The stored delays are assigned to the Time buttons Sets of delays or Sets of time intervals which are no longer required can be deleted e Open the list box and select the required set 03 06 B 45 0019 e 4 99 Carl Zeiss OPERATION Acquire Menu LSM 5 LIVE LSM 5 LIVE DuoScan e Click on the Delete button The set will be removed The Time buttons can also be activated via keys 1 to 4 of the Trigger Control e Click on the required Time button e Open the Trigger in list box with a click on the arrow button e Choose one of the trigger keys 1 to 4 e g Trigger3 It is also possible to trigger an out signal via Trigger Control e Click on the required Time button e Open the Trigger out list box with a click on the arrow button e Choose one of the trigger keys 1 to 4 e g Trigger3 In this example the relevant Time button Is activated on pressing key 3 of the Trigger Control and an out signal is given at the same time 3s The delays or time intervals can be changed online with a click on another Time button The new delay will b
332. in the Maintain subordinate toolbar of the main menu Position Speed Plan Neofluar 1070 3 a H _ _ _ J ters Plan Neotluar 20 05 The Objective Control window appears on d _ T stars the screen The Focus Speed has to be aE activated in the Objective Control window Ko EE The focusing speed of the relevant objective Plan Apochromat 63 1 4 Oil l dH TE can be selected by using either the slider or the input box in 40 steps Empty position E ofa Steps Empty position iens Fig 4 224 Focus Speed window T Objective Control 4 10 2 3 Parfocality Correction Object Focus Speed SE l Oee Foowsoeea dees The partocal setting is performed via screen dialogs in successive panels Parfocal Correction Activation Notice When parfocal correction is active the moving Click on the Parfocal Correction button stage can dammage your objective The Parfocal Adjustment panel appears Parfocal conection active e Start the setting with the objective of the highest magnification factor reference ee eee a objective Proceed in accordance with the Eee displayed instructions e Click on Start Parfocal Correction Adjustment The next dialog is displayed in the Parfocal Adjustment panel e Focus on your slide object Start ert Fig 4 225 Objective Control window e Perform these steps for each objective e Click on the Next step button 4
333. indow e Select the image for the projection operation from the image selection box 03 06 B 45 0019 e 4 155 OPERATION LSM 5 LIVE Carl Zeiss 3D View Menu LSM 5 LIVE DuoScan 4 7 2 3 Projection Panel e On the Projection panel set the parameters needed for the animation Turning Axis First Angle Number Projections and Difference Angle in the Projection tab and the Mode parameters in the Transparency tab 1 Projection tab Turning Axis X Y Z Selects the axis about which the data package is to be rotated First Angle Rotation angle in degrees Number Number of projections for a sequence variable from O to 100 Projections Difference Angle Angle increment of a sequence f The number keys permit the direct selection of preset values for Number Images and Difference Angle If the Panorama button is pressed a panorama sequence of the image series is computed Projection Transparency Turning Axis Cx fy zZ First Angle 7 af 1 478 Number Projections 16 As D j A 45 18 J Difference Angle e of Panorama Fig 4 150 Projection tab 4 156 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan 3D View Menu Carl Zeiss 2 Transparency tab Mode Maximum The color is defined by the z position of the brightness value Mode Transparent The transparent projection is built up from the rear to the front The color is defined by the z position at which the original was last higher th
334. indow of all beam splitters available e To select a beam splitter click on the respective line of the list The selected beam splitter moves into the beam path e Proceed accordingly to contigure the NFT secondary dichroic beam splitters Z 4 56 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 3 Beam path Emission filter Configuration Control e On the Beam Path and Channel Assignment panel click on the T emission filter symbol j i i Multi Track Rati This opens a graphical pop up window of all segete manae o E available emission filters e g BP for band Sean Pah and Channel Aignmeni Spectra pass or LP for long pass with their USM Ducsean eee wavelengths Channel Mode e To select an emission filter click on the respective filter in the pop up window S NFT 490 The emission filter selected moves into the Contig beam path in front of the PMT photomultiplier e Depending on the application it may be necessary to insert additional mirrors secondary rss Z dichroic beam splitters or neutral glass filters between the Achrogate main beam splitter and Speuaner the PMT photomultiplier To select these components click on the respective Z symbols L For channels 1 and 2 it is possible to change the filters directly on the LSM 5 LIVE scan module see CHAPTER ANNEX Filter Change in the Detection Beam Path Fig 4 39 Configuration Control
335. ined for this purpose by the manufacturer Damaged units or parts may only be repaired or maintained by the responsible service agency During maintenance or repair carried out by the service personnel the customer is requested to stand aside and wear a pair of laser satety goggles if needed A Before opening the housing of the halogen lamp switch off all laser units Care operations that may be carried out by operating staff are limited to cleaning painted and glass R e cleaning the instrument make sure the main power supply is disconnected e Cleaning painted surfaces To do this use a clean cloth that has been moistened in a mixture of water and some detergent do not use any solvent however Dry with a lint free cloth e Cleaning glass surfaces Glass surfaces that have become soiled or which are marked with fingerprints may be rubbed with a clean optical cleaning cloth If soiling is persistent dip the optical cleaning cloth into a mixture of distilled water and a small quantity of detergent To complete cleaning lightly breathe on the glass surface and rub it dry with a clean cloth Lint or dust is best removed with a clean brush e Make sure that no cleaning liquid penetrates into the system e Dust filters in the ventilation entries of the system electronic rack have to be replaced every 6 month For replacement please contact your local service representative 03 06 B 45 0019 e 1 19 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl
336. ion The Image Display window appears Starts acquisition of a series of images The Image Display window appears Defines a ROI for camera acquisition in the Image Display window Note that this is just a Crop function while the whole sample is illuminated Rotation of the ROI is not possible Shows the acquisition parameters in the Image Display window Close the Scan Control window B 45 0019 e 4 89 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 5 Edit ROI Region Of Interest A scan image allows certain areas ROIs to be defined Definition and activation of the ROIs for the analysis is performed in the Edit ROI window 4 5 5 1 Open Close the Edit ROI Window e Click on the Edit ROI button in the Acquire subordinate toolbar of the Main menu The Edit ROI window appears on the screen and the ROIs defined last are visible in the Image Display window e Click on the Close button in the Edit ROI window The Edit ROI window is closed and the ROIs disappear from the Image Display window D Fi Fiia aaa bh Bain array Fikia d al FD Heri Minil Jireh E Fig 4 74 Edit ROI window and Image Display window with ROIs When Edit ROI is activated and the first ROI is drawn in the Image Display window the Use ROI is activated automatically 4 5 5 2 Function Description The following functions are available on the right hand side of the Edit ROI window Close button The Edit ROI window is closed R
337. ions can be performed on activation of the buttons in the Overlay toolbar left hand side Xi Arrow selection button Activation of the mouse button for selection resizing or movement of an overlay element in the Image Display window Resizing Click on the handle and hold down the mouse button drag the handle release the mouse button Movement Click on the line and hold down the mouse button move the entire element release the mouse button rd Line button Creation of a straight line in the Image Display window Click and hold down the mouse button draw a line in any required direction release the mouse button to end the procedure m Rectangle button Creation of a rectangle in the Image Display window Click and hold down the mouse button draw a rectangle in any required direction release the mouse button to end the procedure 4 34 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss oI Closed polyline button Creation of a closed polyline figure in the Image Display window The first click sets the starting point each additional click adds a further line a click with the right mouse button closes the figure and ends the procedure gt Open polyline button Creation of an open polyline figure in the Image Display window The first click sets the starting point each additional click adds a further line a click with the right mouse button ends the procedure o Ellipse button C
338. is set up in the factory for every objective belonging to the system This is done together with the Basic Calibration Without running Calibration Matrix the positions of the collimators and BSC Y should be OK This is due to the Basic Calibration but the results are not fully optimized Installation of the Macro If not already done copy the required Tiles to the following destinations Filename pestinaton KolliMatic lvb C AIM Macros PotentialKollimaticConfig ini C AIM Bin KolliMatic_Instructions pdf C AIM Macros 74 Macro Control Starting the Macro Edit Macro Assign Macro to Button 2 e Start the LSM software and check first if all prerequisites have been fulfilled see Paragraph 1 Prerequisites Macros Froject C AIM_35 Macros KolliMatic vb e Put the appropriate calibration slide depending on current objective immersion or non immersion onto the microscope stage e Activate all lasers and using the and focus with Delete the 488 nm line start scanning and focus onto the beads Pent e ia deen e Put the appropriate calibration slide depending on current objective immersion or non immersion on the microscope stage start single scan e Use the Macro Menu of the main LSM software to Load and Run the macro KolliMatic See Fig 4 175 Fig 4 175 Macro Control Window of the main Please wait a few seconds the macro will set up LSM Software beam path and all necessary settings automatically
339. ition on passing that action block 03 06 OPERATION Macro Menu LSM 5 LIVE LSM 5 LIVE DuoScan Carl Zeiss 03 06 e Now the first complete scan has been performed and the image is stored For the next cycle the variable Index has to get another value to make sure the numbering of the images works Using the Assignments action block the Variable Index is chosen and the value assigned is Index 1 Such the variable will get the transient value 2 for the next cycle the value 3 for the over next cycle and so on The number of repeats can be defined using a Decision block and use the value of the variable as base for the decision By defining that if the Expression of the variable is larger than 5 and not connecting the output arrow marked with yes to any other block the macro will stop after the acquisition of five images Alternatively one can use at this part of the program flow the Repeat block and define the number of repeats to be performed which would be 5 in this case The first scan is already counted Property Value Inder Indes 1 Fig 4 197 Property list of the Assignments action block Property Value Indes gt 5 Fig 4 198 Property list of the Decision action block B 45 0019 e 4 197 OPERATION LSM 5 LIVE Carl Zeiss Options Menu LSM 5 LIVE DuoScan 4 9 Options Menu The Options menu permits performance of the following functions Display of a current list of dyes with preferr
340. itioned at any XYZ coordinate of the Z Stack The position of section planes can be changed in various ways By moving the sliders on the Orthogonal Sections toolbar Xand Y settings may range from 1 up to the maximum number of pixels scanned in the example shown 512 Z settings may range from 1 to a maximum of n with n standing for the number of slices produced in the stack By directly entering the relevant number value in the X Y or Z input box and pressing the Enter key If you move the cursor into the Image Display window it changes into a crosslines symbol T By dragging this symbol with the mouse you can position the XZ and YZ section planes to any point of intersection with the XY plane A click with the left mouse button places the intersection to the desired position If you move the crosslines symbol P onto the intersection of the red and green section planes it changes into the a symbol If you now press the left mouse button and keep it pressed you can reposition both section planes simultaneously If you move the crosslines symbol y onto the green section plane it changes into the F symbol If you now press the left mouse button and keep it pressed you can reposition the green XZ section plane You can reposition the red YZ plane in the same way using the Ji symbol The result of an orthogonal section is visible at the image margin Section of the XZ plane green line through the stack abo
341. jecting part of the topography 3 To use the Fill Level function load the Profiles 3D display mode containing the Glowscale palette or activate No Palette to obtain optimum display e f the Diagram function Curve of tp is also activated a red marker line shows the position of the height level in the percentage of contact area curve Farh S12 on 512 of TS Ichra ll Fig 4 298 Image Display window Topography display 3D Volume 4 304 B 45 0019 e alaj xi 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Parameters The following parameters are calculated Z height level selectable with the Z Threshold and Fill Level sliders The setting of this value influences the following parameters Vm z material volume above chosen height level Vv z void volume below chosen height level Smr z material volume ratio Sp 2 5 E Ve Svr z void volume ratio so Va Au surface bearing area of the topography at Z projection area of those parts which are situated above chosen height level Smr surface bearing area ratio of the topography at Z percentage of contact area Au x y 100 Sda true surface sum of all triangles formed by adjacent data points of the surface reconstruction Sdr developed surface area ratio surface areaij projected areaij projected areaij 100 projected area x y The percentage of the 3D surface a
342. k is displayed by a xz image Continuous imaging of the xz stack is also possible 4 82 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Hyperfine Z Sectioning Mark First Last tab Z Sechoning Mark First Last The determination of the optimum stack size IS ne Pe ee eae ae eae Hum 5I 90 qj 1 b performed here Via focusing during a continuous i ae ne _ 4 HOR ae ea A gt nberyval pm scan e Click on the XYcont button f Mark First 34 19 um keep Slice A continuous XY scan of the set focus Keep interval 053 um position will be performed If you have reduced the scan speed or have Fig 4 66 Mark First Last tab set Image averaging you should use the fast scanning mode to find the lowest and highest points of focus These settings are made under Mode in the Scan Control menu or directly via the FAST XY button e Use the manual focusing drive or the Stage and Focus Control window see Stage page 4 114 to focus on the upper position of the specimen area where the Z Stack is to start e Click on the Mark First button to set the upper position of the Z Stack e Then focus on the lower specimen area where the recording of the Z Stack Is to end e Click on the Mark Last button to set this lower position e Keep Slice enables the Num Slices slider which allows to vary the number of slices The limits of the Z Stack remain constant the interval is match
343. k on the VIS button for direct observation e Click on the Micro button in the Acquire subordinate toolbar to open the 5 Microscope Control window of the used microscope ICTO The Microscope Control window appears Fig 10 6 03 06 Selecting an objective e Open the graphical pop up menu by clicking on the Objective button Fig 10 e Click on the objective you want to select The selected objective will automatically move into the beam path Focussing the microscope for transmitted light e Open the graphical pop up menu by clicking on the Transmitted Light button Fig 11 e Click on the On button Set the intensity of the Halogen illuminator using the slider e Click on Close to close the pop up menu e Place specimen on microscope stage The cover slip must be facing up e Use the focusing drive of the microscope to focus the required object plane e Select specimen detail by moving the stage in X and Y using the XY stage fine motion control Setting the microscope for reflected light e Click on the Reflected Light button to open the shutter of the HBO 100 mercury lamp e Click on the Reflector button and select the desired filter set by clicking on it Storing the microscope settings Microscope settings can be stored and up to 8 buttons assigned for fast retrieval and adjustment using the Microscope Settings panel The Store button permits existing microscope configurations to be stored under any
344. ken into consideration for the animation Can be changed during automatic animation End slider The setting of the End slider limits the number of slices to be used for the animation Subsequent slices are not taken into consideration for the animation Can be changed during automatic animation Starts the forward motion of the automatic animation After the last slice has been passed restart is made at the first slice Starts backward motion of the automatic animation After the first slice has been passed restart is made at the last slice Starts the combined forward backward motion of the automatic animation i e when the last slice has been reached the backward motion is activated and the forward motion is activated again on reaching the first slice Stops the automatic animation Move to the first slice After each click on this button backward motion is made by the number of slices set under Increment After each click on this button forward motion is made by the number of slices set under Increment Move to the last slice Speed1 Speed2 buttons sliders Selection between two speeds change of the relevant speed via slider or input box Increment slider Reduction of the slices to be displayed by selecting an increment n step width of slices to be taken into consideration for the animation If n 3 for example only every third slice of the stack will be displayed during the animation B 45 0019 e 03 06 L
345. ks panel in the Name column is deleted Store Apply button Opens the Track Configurations window A selected track defined in a Recording Configuration can also be stored as a single track for single tracking applications Also it s possible to load a single track in a multitracking configuration A click on this arrow button will move the selected track highlighted in blue one position upwards in the list box A click on this arrow button will move the selected track highlighted in blue one position downwards in the list box i 03 06 B 45 0019 e 4 61 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan When adding new tracks the following sequence should be followed e Add a track by clicking on the Add Track button e Determine the configuration of the track in the Beam Path and Channel Assignment panel or select an existing one via the Store Apply Single Track button of the List of Tracks panel e Store the name of a track configuration defined via the Store Apply button of the List of Tracks panel The new track name will then be displayed in the List of Tracks panel If this way of storing is performed the created track will also be available as a single track and can therefore also be activated individually e Add the next track via the Add Track button and then configure and store it again The name of a track can also be changed directly in the List of Tracks panel In that case however the edited t
346. l me el w Contig LSM DuoScan YA A car T 488 30 Z Specimen g Of OOS Fig 4 34 Configuration Control window Single Track activated 4 Detection Spectra amp Laser Lines 488 FITC x SUE cm m mm Ti m m 00 Fig 4 35 Detection Spectra amp Laser Lines window LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 3 1 Open Close the Configuration Control Window e Click on the Config button in the Acquire Subordinate toolbar The Configuration Control window is opened with the display last selected rs The Beam Path and Channel Assignment panel differs according to the hardware configuration supplied e Click on the Close button to quit the Configuration Control window 4 5 3 2 Spectra Button The Spectra button opens the Detection Spectra amp Laser Lines Help74 window This window displays the laser wavelengths activated for excitation as colored vertical lines and the activated channels as colored horizontal bars The color of the bar corresponds to the one as Signed to the relevant channel Non active chan nels receive a gray bar over the entire spectral range The length and position of the bar corresponds to the emitted spectral range which is overlaid with the filter and beam splitters selected in the Con figuration Control window or number of selected channels of the META detector e Click on the Spectra button to open the Detection Spectra amp La
347. l 4 1 1 Software The LSM 5 software Version 4 0 controls the microscope the scanning and laser modules tools filters stand Axioset and the image acquisition process displays and analyzes the images It is based on the network capable graphic 32 bit Microsoft WINDOWS XP operating system Portions Copyright 1981 2001 Microsoft Corporation All rights reserved fs The installation of the software for the LSM 5 LIVE and the basic settings of the equipment components are carried out by Carl Zeiss service staff This job includes the creation of a customized software configuration in line with the specific hardware components of the customer s microscope system The LSM 5 software is menu controlled and normally uses its own windows for the activation of the various functions within these windows further submenus panels can be displayed and removed fr The screen layout used in this manual is based on the former Windows standard layout and may vary from yours Images of the specimens to be examined created by scanning are displayed in separate Image Display windows Theoretically the number of simultaneously opened windows for software operation or image display is unlimited but should not be too excessive so that an overview Is still possible Identical functions e g Laser Control can be performed in several software windows Changes made by the software are recorded immediately and are automatically
348. l pop up menu The shutter is switched on and off When using the X Cite 120 additional controls are available See below for description B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Recording of microscope settings 2x Microscope Settings The upper part of the Axiovert Control window shows the recording functionality of microscope Apply Store Delete _ Assign Button configurations a ee ee Complete microscope configurations can be created and applied Clase The Store button permits existing microscope configurations to be stored under any name The Apply button permits existing stored cr DU Q microscope configurations to be loaded Transmitted Light Transmitted Light The Delete button permits existing microscope configurations to be deleted 4 o Coe The Assign button permits the assignment of a On 3200K microscope configuration to a button Mirror Load a microscope configuration o Tube Lens Lens LSM An existing microscope configuration can be loaded as follows e Click on the arrow button This opens a list box of all stored microscope configurations Fig 4 31 Axiovert 200 Control window e Browse through the microscope configurations by clicking or use the scroll bar at the side of the list box e Click on the desired microscope configuration The selected microscope configuration Is shown in the first line of the Micr
349. l time cintrol unit 03 06 B 45 0019 e 7 11 ANNEX LSM 5 LIVE Carl Zeiss Specification of Trigger Interface LSM 5 LIVE LSM 5 LIVE DuoScan Type Voltage Range TTL signal level 3 3 V CMOS low power consumption 5 0 V tolerant input output for interfacing with 5 V logic Load Output lt 50 mA internal serial 68 ohm resistor Input 4 7 kOhm input impedance internal 4 7 kOhm pullup to 3 3 V Trigger pulse description Signal output Low level lt 0 4 V high level gt 2 8 V Slew rate 10 ns V Signal input Low level lt 0 8 V high level gt 2 0 V Falling edge force interrupt Pulse width to detect signal gt 50 ns Caution Never apply more than 5 V or negative voltages to avoid any damage In and outputs are not galvanically decoupled Therefore proper measures for galvanic decoupling of external devices have to be taken opto coupler etc Hint Use the Zeiss Trigger Box 1315 620 for immediate use of the User Port 4Amomentary on switches 2 BNC connectors including optional signal inversion for input 8 leds and 2 BNC connectors for output 7 12 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX AxioCam High Resolution Digital Cameras Carl Zeiss 7 8 AxioCam High Resolution Digital Cameras 7 8 1 Microscopy Camera AxioCam MRm Rev 2 High resolution microscopy camera AxioCam MRm Rev 2 D Cat No 000000 0445 554 Mid Range Monochrome in
350. lated and shown The final signal intensity in the analyzed ROIs following recovery 10 of the fitted curve 4 148 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss The amplitude of the fitted curve which equals the mobile fraction 11 mobile fraction The titted parameter T1 seconds The rate constant for the exchange of molecules between the bleached region and the surrounding area K per second The part of the immobile fraction of the protein I delta immobile fraction Show Table opens a further table beneath the results It shows all intensity values being corrected for background intensity and intensity loss of the reference region The values can be saved as a text Tile Save Table or copied into Excel via clipboard Copy Table The data can be normalized optionally when marking the checkbox Normalize in the Kinetic Analysis Diagram toolbar mes The calculation of the parameters is based on the same ROIs unless other ROIs or moved ROIs are selected again The Kinetic Display is always available once the analysis has been performed The analysis is not stored with the image fs Note that this modeling is a very basic approach to your experiment It offers a first hint on the possible presence of only one or in case of a bad fit more than one mobile fractions of the labeled protein within the cell or cell compartment examined a Ind 5 half SS The half time
351. lected channel for the scanning procedure by Chl assigning an existing color icon or defining a new one Deactivation of the channel via deactivation of the check box For the configuration of the beam path please refer to the application specific configurations depending on the used dyes and markers and the existing instrument configuration e g module LSM 5 LIVE 1 channel biomedical configurations VarioOne GB 488 532 635 nm 1 channel FL listed in the ANNEX of the printed manual The assignment of the numbered emission filters 1 2 NFT secondary dichroic beam splitters and Achrogate main beam splitters in the Beam Path and Channel Assignment panel is shown in the Configuration Control window Fig 4 38 The numbers of the emission filters correspond to those of the channels lying behind PMT photomultipliers 03 06 B 45 0019 e 4 55 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan u Configuration Contral Emission Filter 1 Beam Path and Channel Assignment ih _ LSM DucSean LSH SLIVE Emission Filter 2 hess mm oe a EP 415420 seggre m Fa By asin mea 7 Specimen Achrogate Q Of OO Fig 4 38 Configuration Control window 2 Beam path Achrogate main beam splitters and NFT secondary dichroic beam splitters e On the Beam Path and Channel Assignment panel click on the Achrogate main beam splitters see Fig 4 38 This opens a graphical pop up w
352. lest frame or ROI fixed size or fitted to the largest frame can be defined by the user CopyPasteOverlays lvb CopyPasteRoi lvb Distance Ivb DivideThroughReferencelmage vb ExcitationFingerprinting lvb Copies actual overlay drawing into the clipboard and pastes the drawing into a selected image window Copies drawing element of overlay into clipboard and pastes it into other selected windows Example macro tor measurement divide complete time series through a single image part of the series duplicate a single image or part of a time series to this series Automated generation of excitation lambda stacks for generating excitation spectra which can be used for linear unmixing to separate overlapping dyes Any detector can be used also NDDs FastModeSwitch vb FileExport vb FRET plus lvb HotKey v KSPlastv lvb Kollimatic lvbo Lambdatrans vb 03 06 Description as guide tour on the installation CD ROM or DVD Store settings from Scan Control and reuse Exports one or more selected images according to the set file format in one go Exports image intensity values in ASCI format Option Enables the user to perform FRET experiments based on sensitized emission or acceptor photo bleaching depending on the type of LSM including commonly accepted analysis algorithms Shift focus with a button and start Single Scan KS software macro Automatically generates the calibration matrix
353. lges in the contour of the region Thin connections between regions are eliminated broken borders between regions are connected and small regions disappear The opposite operation first dilation then erosion is called closing Concave bulges in the contours of regions are filled in connections are formed between adjacent regions The following example illustrates the operations Open and Close in two dimensions Open Erosion Dilation ole Close Dilation Erosion Fig 6 18 Fig 6 19 The cube cross shape was used for the operations shown 6 28 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Morphology Erode This function erodes structures in an image sequence HH Horphology Erode Dilate Open Close Input i all 1 Output E Hew Shape Be Count Poo w 11 0K Cancel W Grey Morphology Fig 6 20 In the Morphology dialog window the tab sheet Erode must be selected Erosion makes bright regions smaller on a dark background It also results in separation of thin connections between regions Small regions disappear entirely If Input is a multichannel sequence any number and combination of channels can be selected Output will only get the selected channels as results The Input image sequence is eroded Count times with the shape Shape The Count scroll bar determines the number of recursive operations The followin
354. lick in the image and hold down the mouse button The perspective is changed by moving the mouse button in horizontal or vertical direction 4 266 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 22 2 Setting via Scrollbars e Move the horizontal scrollbar to rotate the image around the vertical axis The rotation angle is displayed in the yellow display box e Move the 3 left vertical scrollbar to rotate the image around the horizontal axis The rotation angle is displayed in the yellow display box The intensity scale can be varied by the scroll bar on the right hand side of the Image Display window e Moving the J right vertical scrollbar enables you to expand or to compress the intensity scale of the image while the expansion of this intensity axis ranges between 10 and 100 of the X scale size Convallaria2 AIM Display Pseudo 3D Profile Grid Filled og Rainbow Six Step R eady 512 x 512 x 20 3 channels 8 bit Fig 4 270 Image Display window 2 5 D display The Pseudo 3D toolbar contains the following function elements Profile button Profile display vertical polygon display Setting of the Profile Distance between 1 and 20 using the slider Grid button Grid display horizontal grid display Setting of the Grid Distance between 1 and 20 using the slider Filled button Color diagram filled 3D diagram Selection betwee
355. lick on the Delete button e Close the window by clicking on Close 4 54 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 3 4 Settings for Single Track in the Channel Mode The settings of the beam path for the scanning procedure with regard to the main beam splitter Achrogate secondary dichroic beam splitter NFT emission filters EM to be used and the assignment of channels excitation wavelengths and laser intensities are performed in the Beam Path and Channel Assignment panel The setting can be performed manually or by using existing track configurations e Click on the Single Track button unless it has already been activated The Configuration Control window for single tracking is displayed 1 Beam Path and Channel Assignment panel The Beam Path and Channel Assignment panel displays the selected track configuration which is used for the scan procedure You can change the settings of this panel using the following function elements Activation deactivation of the excitation wavelengths check box and setting of Excitation excitation intensities slider Open the Laser Control window via the Laser button Selection of the main beam splitter Achrogate or secondary dichroic beam spliter NFT position through selection trom the relevant list box Selection of an emission filter through selection from the relevant list box i ai Activation deactivation of the se
356. light path and the internal shutter of the lamp Both shutters are closed automatically when switching to LSM mode Opens and closes the shutter in the reflected light path independent of the status of the internal lamp shutter and the attenuation Sets the light via input box or slider Indicates the total time the lamp has been in use 3 The aperture setting on the condensor of the Axio Imager Z1 is performed in fixed steps 4 5 2 3 Microscope Control Window for Axiovert 200 M Transmitted Light button Condensor button Objective button Reflector button Tube Lens button Reflected Light button 4 46 Transmitted light is switched on off via ON button in the Transmitted Light frame setting of light intensity can be varied via input box or slider 3200 K color temperature for photo documentation can be switched on via 3200 K button in the Transmitted Light frame The transmission light control potentiometer on the stand is disabled via the Remote button By clicking on the Close button the Transmitted Light frame Is closed Numerical aperture of the condensor is set via input box or slider Turret position selected from graphical pop up menu only for motorized condensors By clicking on the Close button the Condensor frame is closed Objective can be selected via graphical pop up menu Push and click reflector cube can be selected via graphical pop up menu Push and click tube lens can be selected via graphica
357. list box The selected microscope configuration is shown in the first line of the Microscope Configurations list box e Click on the Apply button e Click on the Close button to close the microscope window f Only those microscope settings which are encoded and motorized can be reloaded Store a Microscope Configuration A newly created or changed microscope configuration can be stored under a new name as follows e Enter the desired name in the first line of the microscope setting list box e Click on the Store button e The actual configuration with the chosen name is added to the microscope settings list e Click on the Close button to close the microscope window 4 42 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Delete a Microscope Configuration A no longer required microscope configuration can be deleted as follows e Select the microscope configuration to be deleted from the microscope configuration list box e Click on the Delete button e Click on the Close button to close the microscope window Assignment of a microscope configuration to Assign Microscope Settings to Button a button Define Buttons A microscope configuration can be assigned to a Button Button button as follows wS e Click on the Assign button Delete Apnly e This opens the Assign Microscope Settings To Button window Fig 4 28 Assign Microscope Settings To Button e Click on the
358. lows to assign a potential error message with a textbox and to decide on the Error where to direct the program flow If no textbox Is assigned the program flow will just continue according to the presence of an error message Choosing a textbox requires the input of the user to continue with the program flow mes With the Release 3 5 this works only for loading images from a database when the images cannot be found because the Index number is not present In all other cases of an error the error message will be displayed and the program stopped This block allows to do image calculation using the pixel intensities of the image The operators for the formulas that can be used are described in the Ratio Dialogue within the Process Menu The formulas set up in the Image calculation window can be copied and pasted into the properties list of the Visual Macro Editor For calculations using two different sources the Calculate block with two possible inputs is used The calculation starts when both inputs have arrived Within program loops the next calculation will start when the inputs have arrived again in the same sequence as the first time The operators for the formulas that can be used are described in the Ratio Dialogue within the Process Menu The formulas set up in the Image calculation window can be copied and pasted into the properties list of the Visual Macro Editor The input image will be copied to a new image format that can be defined b
359. lso see the Time Series Control window Continuous scan not available in Spot Scan mode and Z Stack mode If you select the option Frame for Mode and the option Continuous for Number in the Pixel Depth Scan Direction amp Scan Average panel the Finish button is displayed instead of the Cont button In this case continuous averaging is performed when you have started the scan If you click on the Finish button the scan averaging process is stopped after the scan of the current image has been completed B 45 0019 e 4 69 Carl Zeiss OPERATION LSM 5 LIVE Acquire Menu LSM 5 LIVE DuoScan Additional button in the Line mode Line Sel button Automatically defines a line in the center of the Image Display window Frame for creation of the intensity protile using the mouse the line for the intensity profile can then be positioned anywhere in the Image Display window Additional buttons in the Z Stack mode Start button XYscan button XYcont button Line Sel button Range button 4 70 Triggers the scan of a stack Triggers a single XY scan Triggers continuous XY scan To prepare the Range function a cutline is created in the scanned XY frame to determine the position at which the XZ scan through the specimen is to be produced Using the mouse the line for the XZ scan can be positioned anywhere in the scan frame The cutline can be defined either as a straight line or free shape curve Produces an XZ scan through the s
360. lters OPERATION Process Menu Carl Zeiss User defined Fig 4 120 Filter List and Edit panel Median Tf Filter Close E ay A Click into window Channel Chi v Apply Convallaria Add to List Kemel Size 3x3 Factor 2 5 Divisor fi 2 E Offset fo 3 I Subtract from source Preview Fig 4 121 Filter window User defined filter 03 06 B 45 0019 e 4 131 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan Add Filter to List x e Click on the name of the filter in the Filter List and then on the Remove button The filter will n be deleted Filter Hame Patel e After selection of the required filter click on the Apply button to start the filter procedure Filtering will be performed and displayed in Fig 4 122 Add Filter to List panel the current Image Display window e In the case of images with several channels activate the xy button in the Display image toolbar to display all the channels Each channel must be filtered separately e Use the Save As function to store the newly created image 4 6 7 4 Preview Panel Previ Eie The Preview function allows you to have the result of the Filter operation displayed as a preview Fig 4 123 Preview panel Image e Activate the WI Preview check box with a click of the mouse The Filter Preview Image Display window with the filter result will be displayed e Deactivate the Preview
361. lts in the stored instrument parameters being taken over for active use The track configuration selected before is overwritten cf The optical diagram of the configuration selected appears on the Beam Path and Channel Assignment panel The newly loaded track has been automatically activated for the scanning procedure The Track Configurations window is closed automatically In the Options menu in the function Settings it is possible to define the parameters to be used when applying a track configuration 2 Store a track configuration A newly created or changed track configuration can be stored under a new name as follows e Click on the Config button the Track Configurations window appears on the screen e Enter the desired name in the first line of the Configurations list box e Click on the Store button e Close the window by clicking on Close 03 06 B 45 0019 e 4 53 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan During storage via the Store Apply function all the data of the Beam Path and Channel Assignment and the Detector Gain Ampl Offset Ampl Gain and Data Depth 8 12 Bit scan parameters of the current track single tracking will be stored 3 Delete a track configuration A no longer required track configuration can be deleted as follows e Click on the Config button the Track Configurations window appears on the screen e Select the configuration to be deleted from the Configurations list box e C
362. luding its original accessories and compatible accessories from other manufacturers may only be used for the purposes and microscopy techniques described in this manual intended use In the Operating Manual read the chapter Safety Instructions carefully before starting operation Follow the safety instructions described in the operating manual of the microscope and HBO 100 mercury lamp 2 03 06 Starting the System Switching on the LSM system Set the MAIN SWITCH on the system rack to ON position Fig 1 e When set to ON the System PC_ switch provides power to the microscope and the computer This allows to use the microscope and the computer without running the LSM Software Fig 1 e To switch on the system completely put the Components switch to ON Insert the key into the key operated LASER switch and switch also to ON Now the complete system is ready to be initialized with the LSM Software Fig 1 System rack Switching on the HBO 100 mercury lamp e Switch on the HBO 100 mercury lamp via the switch of the power supply see operating manual of the mercury lamp or microscope Fig 2 HBO 100 power supply 03 06 3 Starting the LSM 5 software program e Double click the LSM 5 LIVE icon on the desktop of WINDOWS to start the LSM 5 software program The LSM 5 LIVE Switchboard window appears on the screen Fig 3 5 Switchboard r Fig 3 LSM 5 LIVE Switchboard menu e
363. lues outside the defined interval will be segmented If the option Binary is selected then all grey values in the range trom Low to High will be set to white grey value 255 4095 in the Output image sequence while all others will be set to black grey value 0 If the option is not selected the grey values within the selected interval remain unchanged while those outside the range will be set to black 03 06 B 45 0019 e 6 35 Carl Zeiss Parameters 6 36 Input Output Colour Binary Invert 3D FOR LSM LSM 5 LIVE Functions LSM 5 LIVE DuoScan Input image sequence Resulting image sequence Green Selected interval is displayed in green Blue Red Grey values below the selected interval are displayed in blue grey values above in red O Selected voxels retain the original grey value 1 Selected voxels are set to grey value 255 4095 the rest to grey value O O Grey values inside the selected interval are segmented 1 Grey values outside the selected interval are segmented Low grey value threshold Center of threshold interval High grey value threshold B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Boolean And This function carries out a bit by bit And calculation for the image sequences Input 1 and Input 2 Boolean And Orl sior Not Mask Input 1 Moo Alf 1 Input 2 pooo A Output Booo Hew Fig 6 26 The And tab sheet of the Boolean dialog window
364. macro function permits the recording running and editing of command sequences and their allocation to buttons in the Macro menu e In the Main menu toolbar click on Macro This opens another subordinate toolbar in the Main menu Using the Macro Button optional opens the dialogue for editing and recording Macros based on VBA programming Using the Visual Button optional allows to construct automated work flows using the arrangement of symbols which depict the single steps within a work flow See Visual Macro Editor page 4 189 for further details A LSM 5 LIVE ki E3 File Acquire Process 30 View Macro Options Maintain Window Help Maintain Fig 4 168 Macro menu 4 8 1 Macro Language Visual Basic for Applications called VBA in the following is used as the Macro language This language is well known through its widespread use as Macro language in the Microsoft Word for Windows and Microsoft Excel for Windows products Experience with Microsoft Visual Basic would also be beneficial for macro programming of the LSM 5 LIVE An Integrated Development Environment called IDE in the following is available for the editing and debugging of macros IDE includes an online help program where the VBA language is described in detail Macros are stored in project files One project file can include several macros 03 06 B 45 0019 e 4 167 Carl Zeiss 14 Macro Control New Load Save Recording Rec Ham
365. mages LSM 5 LIVE DuoScan Render Series 2 Start and End mode Pegioction In the Start and End mode the image is Mene 3 a reconstructed between a start position and an end roo a The rotation angles for X Y and Z and the distance zoom can be determined using the sliders Number of Wiews fia 5 1 4 a 1E 32 Bd Load Save The value for Number of View can be varied Angle Angle r Angle 2 Distance nterpolate Fig 4 277 Render Series window e g Start and End mode Render Seres Projection Close Mode Tum around Tum around 7 Start and End Position List A E ime senes Number of views fio 5 4 a 1E A E4 z i Load Add Position Save Feen WeetE Interpolate Light Background Transparency Channel color Surface threshold View angle 3 Position List mode In the Position List mode the image is reconstructed between any required number of interim positions to be determined individually The rotation angles for X Y and Z and the zoom can be determined directly in the image Every required interim position is included in the list of the Render Series window with a click on the Add Position button Clear List permits the contents of the list to be deleted The value for Number of View can be varied e Click on the Apply button calculates a spline along all the defined positions from the list and starts an animation along this
366. mber scans Different 2 Position D Stop Trigger in None Trigger out None Fig 4 95 Settings for Bleach Series TA Bleach Control Settings FRAF Myocandiac Cs 43 MUT Apply Store Delete Bleach after dl Scan Number E number scans Bleach repeat after mo number scans l Scan Humber 10 Different Position 11 33 prm Pe K gt Max Pinel Time 6 40 ps Trigger in None Trigger aut None Bleach Parameter Iterations 50 Me Define Rea Excitation of Bleach Track Different settings for different ROl z RoI fT 2 Line active Transmission 2 hee A M 458mm 100 4 jo C 477mm a1 gt gt a8anm 0 1 gt Bl4nm 0 1 S gt S61 nm 0 1 m gt 633nm 0 1 m gt 800mm 01 Ch Fig 4 96 Bleach Control Window 4 110 OPERATION Acquire Menu LSM 5 LIVE LSM 5 LIVE DuoScan In the upper part of the Bleach Control window the time parameters can be set Check the required boxes and type in the number of the images where the bleach event is required The number has to be lower than the overall number of images choosen in the time series dialogue Start the bleach series with StartB in the Time Series window 4 5 8 Edit Bleach LSM 5 LIVE DuoScan The use of this function permits the setting of bleaching parameters for spot line or frame bleaching 4 5 8 1 Open Close the Edit Bleach Window e Click on th
367. mber states of the European Union which takes care of the appropriate utilization according to the said EU guidelines For details on the disposal and recycling please refer to your relevant Carl Zeiss sales or service organization The product must not be disposed in the household waste or through the municipal disposal organizations In case of resale the seller is obliged to inform the buyer that the product has to be disposed according to the said regulations 1 10 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Protection against diffuse laser excitation Carl Zeiss 1 4 Protection against diffuse laser excitation emitted around the objective and sample Though the radiation levels around the objective and sample is very low comparable to a common laser pointer used for e g talks avoid to approach this area closer than 10 cm with your eye Also limit the time of skin exposure of your hands to the time needed to place the sample and to me chanically set up the acquisition Switch off the scanning while doing such manipulation around the objective and sample In case of scanning don t stare into the beam To further reduce the radiation level of fluorescent emission and excitation light around the microscope Carl Zeiss delivers antiglare screens for all microscopes Axiovert 200M OO0000 1 109 741 Axio Imager Z1 M1 452163 0000 000 Axioskop 2 FS mot 452163 0000 000 if W Fig 1
368. me of the result image sequence If the sequence exists it is overwritten Pressing the button New will generate a new name number The size of the sequential images in Output is determined by the size of the sequential images in Input Number of Views determines the number of reconstructions which should be computed The radio buttons Start and End define which angle settings are currently shown A definition for the angle End is only necessary if Number of Views is higher than 1 If this is true the result sequence will get views from the Start to the End angle definition The other reconstructions are determined through the linearly interpolated intermediate angles The direction of view is determined from the angles as follows The angle Angle Z determines the rotation of the direction of view on the Z axis The angle Angle Y determines the rotation of the direction of view on the Y axis that has been rotated by the angle Angle Z The angle Angle X determines the rotation of the direction of view on an X axis that is rotated by Angle Z and Angle Y Channel defines if the following parameters are valid for All or just for one Defining the thresholds for the channels independently is useful if the grey value boundaries of the objects differ too much in the different channels The thresholds Grey Low and Grey High define the grey value range of the objects The parameter Aperture is a measure of the size of the highlights Small values generate la
369. mechanical knocks and impact of the LSM 5 LIVE components should be avoided We would recommend you to always use the actively vibration damped table The specifications of the stage are obtained only after a heating phase of approx 30 minutes Furthermore the installation conditions for the LSM 5 LIVE system must be observed The maximum reproducibility better than 40 nm tor moving to an absolute position in Z is achieved by always moving to the required position trom below 03 06 B 45 0019 e 7 ANNEX LSM 5 LIVE Carl Zeiss Hints on the Use of the Piezo Fine Focusing Stage LSM 5 LIVE DuoScan Fine focusing is performed mechanically via an inclined position of the stage tongue Therefore the lifting range Z at the location of the image field depends on the position of the piezo stage in relation to the optical axis This means if the user shifts the object on the microscope stage to the right via the piezo stage the lift will be different from the one in the zero position of the stage max 250 um and also trom the one after a shift of the stage to the left If the LSM 5 LIVE system is equipped with a motorized scanning stage this shift is read back to x and the lift is calibrated automatically if the zero position of the piezo stage has been matched to the zero position of the scanning stage via an initialization run For this activate the Stage button of the Acquire toolbar Then position the scanning stage in such a way that the op
370. mory is running low delete some images from the Gallery If the images are needed afterwards they must be saved to disk first Normally all functions produce a new result output image sequence In order to save some memory other image sequences currently presented in the Gallery can be selected as result position The output image is overwritten by entry execution of a function 6 2 User Interface 6 2 1 Introduction This section describes the following main components of the system Main window Main window with the Menu the Tool bar and Gallery All general system functions are located here Gallery Normally several images are required in order to accomplish a particular task These images are displayed in reduced size to provide an overview and facilitate selection This area is located just below the Tool bar re 3D for Zeiss LSM 510 File Edit Process View Measure Windows Help gt Dal SS Ez Le oF fal Used 2 500 MBytes a of ve Fig 6 2 Tool bar This menu shows all image processing functions 03 06 B 45 0019 e 6 3 3D FOR LSM LSM 5 LIVE Carl Zeiss User Interface LSM 5 LIVE DuoScan Display window This window is used to display image sequences Display 1 Xx SEmi fiefs E E E 312 316 43 22 8bit Fig 6 3 Display window Dialog boxes All dialog boxes provide three buttons Pressing the OK button executes the function with the defined parameters and closes the dialog window
371. n Creation of a straight line in the Image Display window Click and hold down the mouse button draw a line in any required direction release the mouse button to end the procedure Rectangle button Creation of a rectangle in the Image Display window Click and hold down the mouse button draw a rectangle in any required direction release the mouse button to end the procedure Closed polyline button Creation of a closed polyline figure in the Image Display window S O The first click sets the starting point each additional click adds a further line a click with the right mouse button closes the figure and ends the procedure Open polyline button Creation of an open polyline figure in the Image Display window N The first click sets the starting point each additional click adds a further line a click with the right mouse button ends the procedure Ellipse button Creation of an ellipse in the Image Display window O The first click sets the center point the displayed line permits the determination of the first dimension the second click sets the first dimension the second dimension and rotation direction can then be determined the third click sets the second dimension and direction and ends the procedure Closed free shape curve button Creation of a closed Bezier figure in the Image Display window w The first click sets the starting point each additional click adds a further line a click with the ri
372. n Functions Carl Zeiss Object Features densitometric Group Name Name Description Mean Densitometric MeanD Densitometric mean value of an object Standard Deviation StdD Standard deviation of the densitometric values of an object Densitometric Minimum MinD Minimum grey value of an object Densitometric Maximum MaxD Maximum grey value of an object Densitometric Automatic Object Measurement Volume Features A measurement feature describes a region characterized by a number e g volume area or a densitometrical statistic The features can be selected on the Object Features and Volume Features tab sheets lt Automatic Object Measurement Available Features Selected Features V alCount Volume Volume percentage Surace area Mean densitometric gt gt Standard deviation densitometric Minimum densitometric Maximum densitometric Select All Remove All Cancel Apply Fig 6 38 The measurement features can be selected individually for each measurement The object features generate a result value for every single object 03 06 B 45 0019 e 6 55 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan The dialog shows two lists One shows the Available Features as groups on the left The other one shows the Selected Features Double clicking on items of the left list will add the Selected Features to the right list Double clicking on an item of the right list will remove this item from
373. n Handling HBO and X Cite Light Sources a For LIVE systems equipped with a rearport mounted scan head be aware that the mirror cube in the reflector turret leads to a strong back reflection of HBO light into the specimen plane and the ocular lens When observing the specimen through the ocular lens the use of the mirror cube position should be avoided The light flash is not harmful but unpleasant The reflex of closing the eyelid is sufficiently protective To avoid this situation an additional filter LP 397 from filter cube 01 48 80 01 can be mounted into the mirror cube which prevents the back reflection of the HBO UV light in the ocular plane 1 10 Physical Dimensions Passively damped anti vibration table Scanning Module LSM 5 LIVE Scanning Module LSM DuoScan Microscope Laser Module LIVE Laser Module UV for DuoScan only System Electronic Rack Plug in unit external laser UV Power supply for Ar UV Cooling unit for Ar UV 1 11 Environmental Requirements 1 Operation specitied performance T 22 C 3 C without interruption 24h a day independently whether system is operated or switched off 2 Operation reduced performance T 10 C to 35 C any conditions different trom 1 and 5 3 Storage less than 16 h T 40 C to 55 C 4 Storage less than 6 h SoD C t0 70L 5 Temperature gradient 0 5 C h 6 Warm up time 1h for high precision and or long term measure ments gt 3 h 7 Relative
374. n any required direction release the mouse button to end the procedure Free angle button Creation of a free angle in the profile diagram Display of the enclosed angle max 180 The first click sets the starting point the second and third clicks define the angle and the end point K o Rectangle button Creation of a rectangle in the profile diagram Display of distance area height and width Click and hold down the mouse button drag the rectangle in any required direction release the mouse button to end the procedure 4 296 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Oo W N ao Sf fw LJ 03 06 Open Polyline button Creation of an open polyline figure in the profile diagram Display of the length of the line figure First click sets the starting point any further click adds another line click with the right mouse button ends the procedure Closed Polyline button Creation of a closed polyline figure in the profile diagram Display of the perimeter of the figure First click sets the starting point each further click adds another line a click with the right mouse button closes the figure and ends the procedure Open free hand curve button Creation of an open Bezier figure in the profile diagram Display of the length of the line figure First click sets the starting point each further click adds another line a click with the right mouse butt
375. n of the respective laser wavelength to the objective used and thus optimum correction for Z aberrations The two simultaneous image acquisition channels usable for fluorescence are ideal for the investigation of multiple fluorescence specimens In the two channels the diameters of the aperture and their XY positions can be optimized and the desired emission filter placed into the beam path by servo motor control In the simultaneous registration of multiple fluorescences identical optical sections can be obtained in each confocal channel This is of importance e g with the FISH method fluorescence in situ hybridization used for genome analysis in cytogenetic studies It is possible to superimpose a multiple fluorescence image on a brightfield differential interference or phase image In addition to the emission filters for all standard and special applications available in motor controlled filter wheels the user can easily install his own emission filters in two of the channels The high NA C APOCHROMAT objectives specially developed for the LSM technique reach the physical limit in resolving power and can be used throughout the 350 700 nm spectral range with the same high quality producing brilliant images A mirror scanner system controlled by Realtime Electronics offers several advantages The large deflection angle of the scanning mirrors allows a wide area to be scanned With a 1 25x objective the object area scanned is 1
376. n or Sum in the Method selection box e Select the desired scan average from the available values 2 4 8 and 16 in the Number selection box or Continues only for Frame average mode LB The greater the number of averages selected for Mean average Method the better the image quality will be the scanning time will be prolonged accordingly Averaging can be performed in different ways depending on whether the Mean or Sum method has been activated If you are using the Mean method the image information is generated by adding up all scans pixel by pixel and then calculating the mean value In the Sum method the pixel values of all scans are only added up without a mean value being calculated To create the image information using the Line average mode each line depending on the setting is scanned 2 4 8 or 16 times during Scan Average and then the average value per pixel is calculated This minimizes noise interference during the scanning procedure If the Frame average mode is used to create the image information the complete frame is scanned 2 4 8 or 16 times depending on the setting The average value is recalculated after each frame scan The Frame average mode also permits continuous averaging 03 06 B 45 0019 e 4 73 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan For this select the Continuous option in the Number selection box If you have selected the Continuous option the Finish button for
377. n the LSM 5 LIVE must be observed Check whether all of the labels shown below are provided on your instrument and contact Carl Zeiss Germany or one of the service agencies if you should discover that any of the labels should be missing You will receive a free replacement Description of labels Caution Faults and hazards that might arise during operation which might cause damage to the unit or injury to the user Attention Laser irradiation hazards possible when operating the system Attention High voltage Pull the mains plug before opening the device housing Caution Hot surface Caution UV radiation Caution Fingers can be caught The arrow points to the opening where laser light comes out during operation of the system IE Other labels on the system include one of the above depicted symbols and a detailed description of the handling instructions See also the following drawings of the system parts 03 06 B 45 0019 e 1 3 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss Warning and Information Labels LSM 5 LIVE DuoScan Warning LED is lighting up when laser is on J x Fig 1 1 Warning and information labels on the Axiovert 200 M microscope with the LSM 5 LIVE scanning module 1 4 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Warning and Information Labels Carl Zeiss Warning LED is lighting up when laser is on v SALL Fig 1 2 Warning and information l
378. n the Mono Rainbow and Six Step color palettes Channel list box Permits the selection of a Channel in a multi channel image 03 06 B 45 0019 e 4 267 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 23 Display 3D Image VisArt This optional function allows to reconstruct and display 3 D fluorescence image stacks or time series of frames and stacks trom the Image Display window select from a variety of reconstruction modes The settings of Chan are applied The settings of Zoom Slice Contr and Palette are not applied Click on 3D will display the 3D toolbar Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed I The 3D button can also be used online during scanning e Click on the 3D button The 3D toolbar will be displayed f Convallaria2 AIM x Display 3D go Shadow xy Ax EE ont Split xy Ay Eies Ortho Any View Cut T deal ile ooog ia Beery Basic Gallery Thun Advanced mee Surface Profile Basic PAST BI Advanced 3D Full Res aw Ve Topo Appearance Prey Series Ready 512 x 512 x 20 3 channels 8 bit Fig 4 271 Image Display window 3D display The 3D toolbar contains the following function elements Shadow projection Front button Shadow rendering front view Back button Shadow rendering back view Any
379. n to be displayed 6 44 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Grey High High grey value threshold of the region to be displayed Aperture Measure of the extent of the highlights Reflection Weight of the defuse brightness components in comparison to the highlights Auto Update O Function execution is performed on OK or Apply 1 Function execution for the current angle is performed on any parameter change Show Cube O The wire frame cube is not shown 1 The wire frame cube is shown in the Display window Render Surface Method Description This method displays the surface of structures in the Input sequence shaded as if a light illuminated it The position of the light is behind the view point with parallel rays in the direction of the sequence The input sequence is segmented into object and background by grey value thresholding object voxels are within the grey value range Grey Low to Grey High Each Output pixel corresponds to a point at the surface at which the ray in view direction through the Output pixels hits the surface All rays are parallel The surface normal required for shading in this gradient renderer is the grey value gradient in the Input volume at the surface voxel position It is not the geometric surface normal The grey value gradient is determined trom the grey values in a 3x3x3 cube around the surface voxel by averaging e g the x gradient in y and z direction 4
380. nalysis of Images Carl Zeiss Overlap coefficient overlap coefficient after Manders Manders Verbeek and Aten J Microscopy 169 375 382 1993 gt Ch Ch2 S Ch 5 2 Another parameter used to quantify colocalization in image pairs F Insensitive to differences in signal intensities between the two channels photo bleaching or amplifier settings Value range O 1 0 no colocalization 1 all pixels colocalize 4 13 21 Display Profile 4 13 21 1 Function This function allows to display the intensity distribution of an image along a straight or curved line show the intensity values in table form and copy table to clipboard or save as text Tile show separate profiles for each channel in a multi channel image The settings of Chan Zoom Slice Contr and Palette are applied Click on Profile will display the Profile toolbar Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed e Click on the Profile button The Profile toolbar will be displayed The intensity curves are shown in a graph below the image s e On the Profile toolbar you can select the width and color of the profile line e You can place two markers on the profile line to measure differences in intensities and distances in the XY plane L The Profile button can also be used online during scanning e Click on the Diagr in Imag
381. nction to vary the illumination conditions reflection properties and projection settings of the topography You can either select preset Shading Models or use parameters specifically defined as required The specifically defined parameters can subsequently be stored as a Shading Model and are then available at any time for further use Shading Models can also be deleted if no longer needed Load a Shading Model e Open the context menu with a click of the right mouse button then click on the option Render properties with the left mouse button The 3D Rendering window is opened e Click on the name of the required model in the Shading Model List The parameters are immediately set for the current topography e Click on Close to close the 3D Rendering window again 4 290 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Defining a specific Shading Model 3D Rendering Shading Model List e Open the 3D Rendering window e Click on the Define button Default e Change the parameters of the topography using the appropriate sliders e Save the settings by clicking on the Add to List button The Add Shading Model to List window is displayed Distance Azimuth Elongation e Enter a name for the model and click on OK The model is included in the Shading Model Background List I gt See also Appearance Settings
382. nd movie formats e g tif jpg bmp pcx avi mov etc are supported f When importing series please make sure to select the first image for the representation of the entire series and to select the Image Series option under Image type e Finally save the image in the desired image database via the Save As function e In Processing History this file is marked as imported file 4 32 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss 4 4 6 Export of Images The Export function allows the export of acquired images and images loaded from the image database Select the image to be exported Click on the Export button in the File subordinate toolbar of the Main menu This opens the Export Images and Data window Under Save in select the data medium and the directory to which the image is to be exported Enter a name for the image under File name Select the image format into which the image is to be exported under Image type Single Image with raw data Contents of the Image Display window Full resolution Chose a compression level For some file formats lossless compression or various other compression levels are available The degree of losses for the image quality are listed according to the type of compression Click on the Save button The image is stored on the relevant data medium directory All the usual image and movie formats e g tif jpg bmp pcx avi mov
383. nd the laser will not be damaged 2 16 Laser Module UV for LSM DuoScan UV Single mode polarization preserving Tiber Laser beam attenuation UV AOTF Ar UV Laser 351 364 nm 80 mW 2 12 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE DuoScan System Overview LSM 5 LIVE Material Carl Zeiss 2 17 System Overview LSM 5 LIVE Laser Enterprise II 653 80 mW 351 nm 364 nm UV Laser module Laser module LSM 5 LIVE with output coupling Plug in unit fo external laser Scanning stage 130x85 PIEZO for upright stand XY stage controller PIEZO XY Joystick for stage controller PIEZO LCD TFT flat screen monitor 19 16 10 flat screen monitor 24 Scanning stage DC 120x100 with mounting frame for inverted stand Control computer LSM 5LIVE System table with breadboard Wide 1000x750 mm 1200x950 overall 2 axes control panel Narrow 750x1000 mm 950x1200 overall 03 06 B 45 0019 e 2 15 LSM 5 LIVE SETUP REQUIREMENTS Carl Zeiss System Overview LSM 5 LIVE Material AxioCam HRm AxioCam HRc AxioCam MRm Ae DAA f Y is Microscope stand Axio Imager with TFT monitor and light control mot O 9 Og i i Z LP Axiovert 200 M SP Axiovert 200 M RP AxioCam HRm Several solutions for incubation AxioCam HRc will be offered AxioCam MRm LSM 5 LIVE lt gt AxioCam HRm o AxioCam HRc A
384. ndow 4 6 10 1 Open Close the Channel Shift Window e Click on the Close button to quit the window 03 06 B 45 0019 e 4 135 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan Click into window F ml w ee 4 4 Unnamedd2 Fig 4 129 Image panel Horizontal lo E Vertical E E Channels D Chi Ch3 Fig 4 130 Shift panel 4 6 10 2 Image Panel e Click on the arrow button The image selection box will be opened and all the currently loaded images are displayed in a minimized form e Click on the required image which will then appear in the display box of the image selection box and has been selected for the Shift function L gt You can also use the Click into window button for image selection 4 6 10 3 Shift Panel e Select the channels required for processing in the Shift panel by clicking on the Ch1 or Ch3 buttons A tick will appear in the button when the channels are activated e Use the scrollbar or the 4 and J buttons to select the pixel shift in the horizontal and vertical direction e Click on the Apply button to activate the setting 4 136 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss 4 6 10 4 Preview Panel e f Preview is activated a preview of the shift Pi is shown in a separate Image Display window Fig 4 131 Preview panel The following image shows the result of a pixel shift vi
385. ndow 4 322 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Image Optimization Carl Zeiss Pinel Gepth Scan Deemon amp Scan wera gt RAS SY Pesii Bae 1 chr a Fig 4 317 Scan control and Image Display windows As a rule the first scanned image Pre Scan is not ideal since the photomultiplier is not matched to the light output More often than not the screen image is dull and needs subsequent optimization e Click on the Channels button in the Scan Control window This opens the Channel Settings and Excitation of Track panels The channels used are color highlighted 4 14 2 2 Image Optimization The image optimization processes setting of aperture size Detector Gain Ampl Offset Scanning speed and Average must be carried out separately for each channel used see Single Channel page 4 315 For the optimum setting of the single channels Split xy display must be selected in the Image Display window to enable the direct viewing of the separate images of the relevant channels e Click on the Cont button in the Scan Control window This starts a continuous scan e Click on the Split xy button in the Image Display window toolbar This displays the separate images scanned in the channels and the composite overlay image 03 06 B 45 0019 e 4 323 OPERATION LSM 5 LIVE Carl Zeiss Image Optimization LSM 5 LIVE DuoScan e Now click on the ChL1 button
386. ndow By clicking this button the actual image acquisition is started 4 96 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss ASAD Stop Setter Pane PStop Sores Trigger Time In this panel the parameters for the end of the ose WEEE time series are set and the number of cycles is Number 4 4 e gt determined Trigger out None Fig 4 82 Stop Series panel The following functions are available Manual button The time series is finished manually with a click on the Stop button Trigger button The time series is finished via a trigger signal Time button The time series is finished when the set time has been reached The internal computer time applies as the set time Number input box Determination of the number of images acquired or image stacks for the time arrow keys slider series Time input box Input of the time for the end of the time series Time button activated Trigger in listbox Selection of the trigger keys 1 4 with which the end is to be triggered Trigger button activated Trigger out list box Selection of the trigger keys 1 4 for the out signal e Use the slider near Number to select the images or image stacks for the time series 1 Stop via Trigger To end the Time Series via Trigger Control Trigger Manual Trigger Time button activated first determine the trigger key ieee ne ee EE which is to end the Time Series aie gt Trigger in T
387. nearizing the histogram of the image sequence to equal area fractions in the histogram The areas voxel count multiplied by grey value range of all grey values in the Output histogram are the same This function is used to achieve a better view of an image sequence When Skip Black is checked the grey value O will not be taken into account for linearization Input indicates the sequence to enhance If it is a multichannel sequence a single channel all channels or any number can be selected The Input histogram shows the grey value distribution of the selected channels of the Input image sequence Output defines the range of the result sequence It will get only these channels which are chosen by the Input parameter The grey value intensity resolution will be the same as the one from Input The Output histogram shows the resulting histogram The horizontal axis represents the grey values from O to 255 The vertical axis represents the pixel count The vertical scale of the histogram is set using the scroll bar The units are percentages of the grey value distribution maximum This setting has no influence to the function 03 06 B 45 0019 e 6 23 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan Parameters Image Input image sequence Output Output image sequence SkipBlack O Grey value black is ignored 1 Grey value black is taken into account Input L Lower boundary of grey value range Input Input C Center of grey val
388. nenennneannnnan 6 16 Image OPtlMIZAtION eee 4 319 4 323 Delete Channel 6 16 IMAGE SEQUENCE ccccceeceeeeecesseeeneseeenees 6 2 Edit Changes 6 15 EIA E e E EE reer T A IE E 6 2 P a tit at fataasee a heareeratat eae 6 14 Information of the image c ccceee 4 311 Z nies Rasa Ade do ee a ee nie Interpolation s iieii 4 134 orphology s ssnneesinessiresriresrrerrrrenrnes i Morphology Cl0S ose aiiai 6 32 l n concentra UON soia 4 141 Morphology Dilate cceeecccee ences 6 30 K Morphology Erode axis ari giveeactiivecocnsadues 6 29 Morphology Open rsen i 6 31 KEE ran T 4 146 6 0 lag mers 10 E ia ee ene erat 6 52 Open AGS eiczs ach sctuaeacaserandecnitectctanencdee 6 11 L Fees Sacer cect EET E eae 6 14 cel ale e174 614 eee ae ee eee 6 46 Laser attenuation s tacics ieaawece weeeies Seateunianedsvaes 4 59 7 18 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan PASE COMO ern TE 4 38 HN e E A ca ELEA AEE SE E ET 4 68 Creation of a line oeren 4 87 lowpass MHEN srineenennn aa 4 130 LSM 5 LIVE switchboard 4 325 M MaC Oce ES 4 16 4 167 4 215 editing and debugging 4 172 Main components of the system 068 6 3 Main MENU tictaksedecsetointeeiandteddussauecdttoceusees 4 15 Maii WiINGOW fcc sctact tect entern Ee 6 3 Maintal Ss cre cecenaseteaescclearcusescapaenspesne A 16 4 210 AGT sothaarseati dca tentatabtienkitwe es 4 220 Collimator adjustment
389. ng the objectives motorized is inactivated The nosepiece will be moved down automatically before each motorized objective change b The reflector mount is motorized and provided with the Axiovert 200 M reflector turret The reflector turret has 5 positions One transmitting light position which is identical to the LSM position and four further positions for fluorescence filter sets reflector modules If you want to use more than five conventional fluorescence filter sets it is advisable to use a further reflector turret When changing the reflector turret position you must make sure that the turret will click into position since otherwise the image area will be cut c The stand has a motorized focusing drive fine coarse Sensitivity of the focusing drive is adjusted to the delivered objectives by the manufacturer If you want to use other objectives sensitivity and parfocality can be adjusted via the Axioset program d The stand features an integrated power supply for the internal motors and stand electronics The power supply can be switched on at the right side of the stand External power supply units will be used for the mercury vapor short arc lamp e The analyzer slider for conventional DIC methods will be operated from the right side and is located just below the nosepiece When the rod is pushed in the analyzer is located in the beam path In LSM mode the analyzer must not be located in the beam path analyzer rod must be pull
390. nother subordinate toolbar in the Main menu T LSM 5 LIYE El Ei File Acquire Process 30 View Macro Options Maintain Window EditUI Help F aD View TE Options Maintain Fig 4 24 Acquire menu For preparing and acquiring a scanning image it is recommended to call up and use the tools of the Subordinate toolbar in the following order e Conventional microscope setting e Laser setting e Configuring the optical system for the Scanning Mode e Setting of scan parameters e EditROI permits up to 99 regions within a frame to be defined and analyzed option e TimeSeries permits user specific time series to be selected for the scan procedure e Upon selecting Stage you can set the focus Z coordinate and the Z step size between successive Slices If the optional motorized X Y stage is connected the X and Y positions of the sample can also be selected e The VIS TV and LSM buttons switch the beam path and indicate which beam path has been set in the binocular tube of the microscope VIS for viewing TV for camera observation LSM for laser operation with monitor observation LS For the scanning process the LSM button in the toolbar subordinate to the Acquire item must be activated 03 06 B 45 0019 e 4 37 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Laser Control x 4 5 1 Laser Control TEIA EE The Laser Control panel shows the types Diode 488 100 488mm O OOOO excitation wavelengths and operating stat
391. now recorded at the defined time intervals The result is displayed in the form of the combined Image Display window of the stack and time series 4D Unnamed41 AIM l Ri EATE 5 5 ti Display 0 0 pm T Bi is i 40 pm 2 0 um A B 16 0 pm Ready 51285128584 3 channels 6 bit Display oom 1 4 Palette No Palette Intensity 33 359 22 Chl 30 Cha 46 ChA 64 Fig 4 89 Image Display window of a Z Stack and a Time Series Scan The additional Z Time and Z Time buttons are available in the Gallery toolbar of the Image Display window When you click on the Z button the individual frames of the Z Stack are displayed for the selected Time Slice When you click on the Time button the individual frames of the time series are displayed for the selected Z Slice 4 104 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss For Z Stacks over the time 4D following offline functions will be enlarged Slice Z slider and Time slider Gallery Z Time and Z Time buttons 3D slider for single time index To select the Z or Time Slices use the appropriate sliders which are displayed if the Slice button in the Image Display window has been activated When you click on the Z Time button all individual frames will be displayed 03 06 B 45 0019 e 4 105 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 6 8 Time Series with Mean ROI e Set all
392. nside available Software option 3D Deconvolution available Soft tion Multi Time Seri ilabl e Click on the Close button to close the About HO oar i Control Program 4 0 0 67 WIP d OW Interlace Server 4 0 0 67 User Interface 40 0 6F Control Database 3 05 07 5 Asiovert 200 M Microscope Stand Dummy Focus Dummy Laser Modul Mot implemented Scan Unit Mot implemented Scanning Stage Dummy HFSC1 C44 Not implemented T PMT Mot implemented CAN SCSI Mot implemented DSP Mot implemented Fig 4 235 About LSM 5 LIVE window 4 12 3 FRAP Guide FRAP Guide opens up a SW integrated Guide for FRAP experiments Use the buttons in the Guide to set up your experiment Follow the steps and the descriptions You are also referred to relevant literature and the website of the EAMNET see also Kinetic Analysis Tool optional on page 4 146 03 06 B 45 0019 e 4 223 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 Display and Analysis of Images 4 13 1 Structure of the Image Display Window The Image Display window shows the image or images when they are scanned by any scanning function see Scan control and Time series control or loaded trom the image database see Open database Load or imported by the import function see Import Fasi S02 SiZ 0 Dre Eha Fig 4 236 Image Display window showing a In addition to show images the Image Display single frame image window offers two to
393. nt This makes it easy to chose the ROI for analysis The region should be slightly smaller than the original region that has been bleached The latter is listed in the Mean of ROls list in the image window In the ROI Mean Display three ROls are defined See Additional Display Mode in Time Series page 4 312 for further details ee Be ee ee l f T e a Fig 4 140 Image window displaying the first bleached image of a images series e Open the Kinetic dialogue and chose the image series for analysis e Mark the checkboxes for background and reference region e Press Select Click for the Background Region than click into the ROI number 2 Background region in the image The value of the mean intensity of this region will be subtracted from the intensity values within the ROI to be analyzed e Press Select Click for the Reference Region The intensity values of the ROI to be analyzed will be corrected for changes in intensity calculated for the reference region The correction is done for each time point taking the actual intensity difference in the reference region into account e f no other option is chosen the remaining ROI or ROIs are used for analysis Each ROI is analyzed separately e Chose the Kinetic Model in the pull down list then press Apply The result of the fit is displayed in the table The result can be copied to the clipboard Copy Results or saved as a text Tile Save Results The following values are calcu
394. ny Description 71 Channel Fluorescene Motes User Jappcenter Database MOE O14 magesConvallaria mdb Conyvallaria mdb D AIM GUIDED Guided mdb ONE sample Image Databates BioMed Biohed mdb ONE sample Image Databases M ateral M aterial mdb 0 E sample Image Databases NLO SNLO mdb Fig 4 18 Save Image and Parameter As window e Enter the name of the image in the Name text box e g Conv 7 03 06 B 45 0019 e 4 29 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan e Click on the New MDB button This opens the Create New Database window in which you can create a new image database e g Projections mdb see Create New Image Database page 4 18 After creating the new database Projections mdb AIM window appears Save Image and Parameter As Mame Conv Description f Channel Fluorescence Motes Cancel Oper User fappcenter S MR Database MDB fim Image Datas Ulmann mdb Ullmann mdb CMmage Data D atabase Fel 3_O Lambda Stack mdb Lambda_Stack mdb New C lmage Data Database_Rel 3_O Test Rel 3_O mdb Test Rel 3_O mdb MDB Clmage Data ag der offenen TuerDemo Bilder mdb test mdb test mdb CAlmage Data ag der offenen TuerDemo Bilder mdb Demo Bilder mdb alt i Compress Files M Create New Database Ed ES Create in IS Database _Fel 3_0 c J DE Time Series mdb Lambda Stack mdb LI MultiChannel mdb C Test Rel 3_0 mdb File name Frojecti
395. o this a voxel renderer like the gradient renderer would display only the surface of objects that are defined by grey value thresholds This surface would appear shaded as if illuminated by a light 5 The method can also be applied to visualize pronounced structures within other enclosing structures If the structures have different grey value ranges 6 In this case the Opacity Table is defined as a step Low grey values background have weight O High grey values inside structures have maximum weight 03 06 B 45 0019 e 6 47 Carl Zeiss Parameters 6 48 Input Output Number of Views Angle X Angle Y Angle Z Channel Threshold Ramp Max Opacity Opacity Table Auto Update Show Cube 3D FOR LSM LSM 5 LIVE Functions LSM 5 LIVE DuoScan Input image sequence Resulting image sequence Number of reconstructions to be calculated Angle of rotation on the X axis start position Angle of rotation on the Y axis start position Angle of rotation on the Z axis start position All The following parameters are valid for all channels X The following parameters are valid for the selected channel only Grey value where the opacity starts rising Length of the opacity slope Maximum opacity value Maximum opacity value O Function execution is performed on OK or Apply 1 Function execution is performed on any parameter change O The wire frame cube is not shown 1 The wire frame cube is shown in the Display windo
396. oae ranted ean eran ebee 2 2 2 4 Minimum Space Requirements of the System sessisssiiuessriresrirrerrirreerirrerrrrrerrrrreerrrerrrn 2 3 2 5 Dimensions OVEIVICW ceine a aise ae a E ae 2 3 2 6 SchemaUC OUTING or a yS ea n E E EE 2 4 2 7 Pace RCOUILEMEN TS arugain na a a a ra 2 4 2 7 1 ESIV DUO CITANIA IN E ae a ciev see state uspmeniaheutabegentoeeorius 2 5 2 8 POWER REO UINCMICIN Sonae en dceabaghan uted dined ne queteabanduhactunteigeatastmaibachsnad e S 2 6 2 9 PAY SiGe DIMEN ON aseaalscece tan nas ira datets a ce a suicides ta Daaouey aa ducesepoite O athe tn sateplecheaneesadatenad 2 9 2 10 Dimens oror o aie nals pie Crates eee eee ee oe een enn eee eae ee et a ne ee 2 9 2 11 Environmental REguUremen Genen sashut atentni a E alt atv a O E 2 10 2 12 NA NON a E O EE EE E AEA EE nunca 2 10 2 13 M OSCO DE a E E aie s 2 11 2 14 Sannina Moale LM SEVE or E A EE 2 12 2 15 Laser Modole LIVE sirier Ueda iaia e a 2 12 2 16 Laser Module UV Tor ESM Diostan UV seoce chant os EE E 2 12 2 17 System Overview LSM 5 LIVE a ser Sani exnhabadinactineesabans tered tenet Sent lnech ueuth ad te cuadan dean adam eibebauartnds 2 13 2 18 Ssys em Ovemniew LSM 5 LIVE DUOSCAN aisne alton cust ania aes eweulede 2 15 03 06 B 45 0019 e 2 1 LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE Carl Zeiss Power Requirements LSM 5 LIVE DuoScan 2 LSM 5 LIVE SETUP REQUIREMENTS 2 1 General Remarks The LSM 5 LIVE is a user friendly high performance laser scanning system wi
397. of a LSM 5 LIVE system for optimal confocal aperture and collimator settings Time series alternating between lambda and transmission mode B 45 0019 e 4 173 OPERATION LSM 5 LIVE Carl Zeiss Macro Menu LSM 5 LIVE DuoScan a So O OOOO O o LsmHWAdmin lvb also Button in Direct service hardware access password protected Maintain LsmHWAdminEx vb Calibration service macro password protected LsmTime lvb Triggered Time scan Macro MetaExport vb Export of META image Tiles including all channels as tif bmp ModitySeries lvb Modities Z Stacks and Time Stacks like Rotation of the stacks being mirrored Conversion of time stacks into z stacks and vice versa StitchArtPlus lvo StitchArt Plus macro Option MultiTime lvb Set up of time series experiments including repeated imaging bleaching and autofocusing with defined configurations at multiple locations and for various views at each location Software option OptimizeGDC lvb Optimize the max peak power of fiber coupled Ti Sa lasers Release OptimizeGDCV3_2 Ivb 3 0 3 2 Optimize_Live lvb Temporarily adjusts the contocal aperture position of a LSM 5 LIVE without changing the general calibration values Pixel lvl Displays and stores the mean intensity values of each line of one or more channels of one or more images data trom each line are stored as a txt Tile in the current folder Profile lvb Displays the pixel values along a line RecoveryChameleon vb Imitates recovery of
398. of iterations of the bleaching procedure in the Iterations input box Close e Click on the Define Region button a The Bleach Regionsj Helpi94 window appears Add t The definition of bleach regions corresponds to the Edit ROI function and is performed in the same Interactive ROI Definition Way see Edit ROI page 4 90 Type elie pa Dimension x Y ROIs already defined with Edit ROI are also Rectangle p EJE available in the Bleach Regions window They can 145 i a3 a CREN T m be activated directly modified if required and E stored under a new name Elipse 103 pa f IB ROIs newly defined in the Bleach z Regions window will then also be available in the Hz DJO Edit ROI window e Define the required bleach regions in the scan Fig 4 99 Bleach Regions window Image or use an existing ROI 4 112 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 8 4 Excitation of Bleach Track Panel Excitation of Bleach Track Different setti In the Excitation of Bleach Track panel you can for different BOls sales select the lasers and laser intensities for bleaching a E 2 a Oe The setting of the lasers for the bleaching proce M 458nm 100 M gt dure corresponds to that for the scanning proce dure and must be pertormed accordingly see Laser Control Configuration Control and Scan Control 477nm 0 1 4 gt sanm 01 4 gt
399. of mathematical morphology on image sequences HH Morphology Erode Dilate Open Close Input i all 1 Output CE Mew Shape Be Count Poo w 11 IY Grey Morphology Cancel Apply Fig 6 17 As generalization of the morphology of two dimensional images to three dimensions the structural elements are small volumina Literature Bomans M H hne K H Tiede U Riemer M 3D Segmentation of MR Images of the Head for 3 D Display IEEE Transactions on Medical Imaging 9 1990 177 183 Schiemann T Bomans M Tiede U H hne K H Interactive 3D Segmentation of Tomographic Image Volumes 14 DAGM Symposium Mustererkennung Springer Verlag 1992 73 80 03 06 B 45 0019 e 6 25 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan The input image sequence is analyzed voxel by voxel with a selected shape Shape The voxel to be analyzed is always the central voxel of the shape The shape type determines which neighboring voxels are used to compute the resulting voxel The following structural elements are available for all morphological operations They represent approximated spheres with an increasing radius Sequential image Volume view Cross shape Volume view Cross shape 6 26 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Sequential image Volume view Cube cross shape created through application of cube and cross one after the othe
400. of the Gallery is shown in the Input section The selection of the sequence to save is done by highlighting one of the provided names or by drag and drop from the Gallery Parameters Input Name of the image sequence to be saved Filename Name of the file to be used on disk Save Display As This function saves the current Display window contents to a disk or network drive Save Display As Save ire E Images ce ce File name Display bmp Save as type Hana lye a eels Cancel Fig 6 8 Before the execution of this function any image or image sequence can be selected to be displayed From a multichannel sequence any channel status on or off combination can be defined The colours of the shown channels can be set with the function Set Channel Colour The current zoom factor of the Display window is not taken into account the image is saved without any zoom The image is saved as a true colour image with 24 bit resolution From the Save as Type list box one of the provided formats can be selected Parameters None 03 06 B 45 0019 e 6 13 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan Print This function prints the current Display window contents The standard Windows print dialog is opened Before the execution of this function any image or image sequence can be selected to be displayed From a multichannel sequence any channel status on or off combination can be defined The colours
401. oft cloth from the center to the edge but do not use benzine paint thinner record cleaner or static repellent This can damage the disc Do not place the disc in any place where it is exposed to direct sunlight or high temperatures Backup your data on a regular basis Do not install any other software without talking to your Carl Zeiss representative Never turn your computer off before you have terminated the LSM program and run down the WINDOWS XP operating system Otherwise the program and or data files may get lost B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Notes on Care Maintenance and Service Carl Zeiss 1 13 Notes on Care Maintenance and Service The manufacturer of the unit cannot be held liable for damage resulting from operating errors negli gence or unauthorized tampering with the device system particularly as the result of removal or replacement of parts of the unit or as the result of the use of unsuitable accessories from other manufac turers Any such action will also render all warranty claims null and void and laser satety might no longer be warranted You are well advised to arrange a service agreement with your nearest Carl Zeiss representative to guarantee perfect functioning of the microscope system in the long term Modifications and conversion work on the components of the system must only be carried out by the manufacturer by the service agency or by persons authorized and tra
402. olbar Any changes done with this toolbar are effective immediately e A click on the Gallery button in the Display toolbar not only produces the gallery itself but also the Gallery toolbar with two buttons Data button and Subset button e Clicking on the Data button shows the Z Slice distance the acquisition time or the wavelength or combinations e Clicking on the color selection button below the Data button will open a color selection window allowing you to choose at a click of the mouse in which color the data will be shown in the gallery display e Clicking on the Subset button opens another Sues window entitled Subset in which you can select images of the set of images displayed Start Slice End Slice A stack consisting of the selected images only is generated and displayed Eve nth Sice _ e Select Start Slice and End Slice via the sliders the input box or the Click into window button e Enter a value for n in the Every n th Slice Fig 4 259 Subset window panel e If required activate the Convert 12 bit to 8 bit check box b e Clicking on the Ok button will generate a new Subset of images T Convert 12 bit to 8 bit e Cancel will stop the procedure 03 06 B 45 0019 e 4 251 OPERATION Display and Analysis of Images LSM 5 LIVE Carl Zeiss LSM 5 LIVE DuoScan FS oaaae 73d SD lel Pals Ea scoct Dla Galhey ao ao x El sche dl Dal PB pm ry ree
403. olbars for changing the display parameters and save an image or images see Select toolbar below generating new ways of displaying the data as well as analysis tools see Display toolbar below The Image Display window of the LSM 5 software corresponds to the basic structure of other Microsoft WINDOWS applications The Image Display window can be moved as required within the screen and its vertical horizontal and diagonal size can be matched to the current requirements identical to Microsoft WINDOWS The caption at the top of the Image Display window contains the control menu for the Image Display window identical to Microsoft WINDOWS the name of the displayed image and the Minimize Maximize and Close buttons In the status line at the bottom of the Image Display window the progress bar of a current scanning procedure and the parameters used for image display are shown and updated when changed On the left hand side of the Image Display window an overview of the scan parameter is displayed provided that the Info button of the Display toolbar is activated The Settings function of the Options subordinate toolbar with the Image Display tab some of the functions of the Image Display window toolbars can be activated at the opening of a new Image Display window It is possible to display the Chan Zoom Slice and Overlay image display toolbars immediately on opening an Image Display window The relevant check boxe
404. old ja z Ramp Fig 4 147 Transparency tab 4 154 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 7 1 4 Preview Panel The Preview function permits you to regard the intluence of parameter changes in an Image Display window e After finding the optimum adjustment using the Preview function you have to generate the final version of the image using the Apply button The system then generates a color coded depth map for the selected channel 4 7 2 Projection By means of the Projection function one single projection or a series of projections can be calculated after rotation of the data package about the X Y or Z axis A stack of images must be available 4 7 2 1 Open Close the Projection Window e Click on the Projection button in the 3D View Subordinate toolbar of the Main menu This opens the Projection window e Click on the Close button to quit the window 4 7 2 2 Source Panel OPERATION 3D View Menu Carl Zeiss g Depth Coding Convallaria AlM Ready 512 x 564 3 channels 8 bit o Fig 4 148 Depth Coding image Projection Source Close Click into window ie A Apply Convallaria Stack Projection Transparency Turning Axis s oOo ww First Angle n 1 478 Number Projections 116 z ZS fo zieje x 45 18 3 Difference Angle B 5 Y Panorama VV Preview Slice Fig 4 149 Projection w
405. on The LSM 5 software can be operated using the mouse the PC keyboard or both The operation of the mouse and the keyboard is identical to that of the Microsoft WINDOWS operating system and is therefore not dealt with in detail in this manual If required see the Microsoft manual or online help for relevant information 03 06 B 45 0019 e 4 11 OPERATION LSM 5 LIVE Carl Zeiss Switching on the System LSM 5 LIVE DuoScan 4 2 Switching on the System The LSM 5 LIVE system is equipped with a main power switch located at the front of the System Electronic Rack and two further switches labeled System PC and Components The main switch has to be set to ON to be able to switch on and off the system using the System PC and Components switches The main switch can be used to switch off the complete system with one switch only The electronics to run the computer and the microscope are switched on with the System PC switch The lasers and the scan head are switched on with the Components switch These switches are also accessible via the remote power control switch Fig 4 1 3 Note that the full performance and correct adjustment of all internal elements is achieved after a warm up time of 40 min e When set to ON the REMOTE CONTROL switch labeled System PC provides power to the microscope and the computer This allows to use the microscope and the computer without running the LSM Software 3 The drives for floppy discs and CD DVD of the
406. on txt B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 21 3 Display Mean This function allows to display the intensity time diagram mean intensity in user defined ROIs over time display the intensity time diagram for volumes 3D ROIs within a Z Stack over time use frame time series and frame Z Stack time series as input show the intensity values in table form and copy table to clipboard or save as text file show separate diagrams for each channel in a multi channel image The settings of Chan Zoom Slice Contr and Palette apply Click on Mean will display the Mean of ROIs toolbar Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed To use a similar functionality while scanning use the optional Mean of ROI function with the Time series control e Click on the Mean ROI button The Mean of ROIs image display toolbar will be displayed on the right The used ROIs become visible in the image and the Intensity Time diagram is shown on the left of the Image Display window Unname d16 AIM Select Display Mean of ROIs Zoom ring scan o Type Position Dimension Yh ata ws Vi O 43 152 88 Diagram Bs Be FD Plod Es E or Mono Area f Mean 1 Threshold c Ch3 Ch4 Low pe rc ies 00 01 02 03 04 05 06 OF 08 09 1
407. on or titration calibration compare F with a calibration curve titration calibration or put F values in calibration formula lice Ds ee n Raw Images Sueno ie a a Background and Autofluorescence image Calibration type EOE _ a amt a i Mae aa at Dye n E Eger T Tion R no hi Scaling of calibrated image ins oe Fig 4 136 lon Concentration window 4 142 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss 4 6 12 4 Ratiometric Dyes e Fura 2 Indo SNARF Cameleon Ratiometric Pericam Phluorin e Display fluorescence ratio R over time e Display fluorescence ratio R corrected for background autotluorescence over time e Calculate absolute ion concentrations pixel by pixel via titration calibration known ion concentrations applied to the cells in situ or in solutions In vitro or equation calibration where possible Fura 2 Indo SNARF e Calculation of R eliminates artifacts and uncertainties caused by inhomogenous dye distribution photobleaching may be applied with moving cells 4 6 12 5 Ratiometric Dyes Online Ratio R t1 F1 t1 F2 t1 R t2 F1 t2 F2 t2 Fig 4 137 Ratiometric Dyes Online ratio 03 06 B 45 0019 e 4 143 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 12 6 Ratiometric Dyes Calibration e Subtract background autofluore
408. on ends the procedure Closed free hand curve button Creation of a closed Bezier figure in the profile diagram Display of the length of the line figure First click sets the starting point each further click adds another line a click with the right mouse button closes the figure and ends the procedure Ellipse button Creation of an ellipse in the profile diagram Display of the area First click sets the center point the displayed line permits the determination of the first dimension second click sets the first dimension the second dimension and rotation direction can now be determined third click sets the second dimension and direction and ends the procedure Circle button Creation of a circle in the profile diagram Display of radius and area Clicking three times to define 3 points on the profile A circle fit is automatically applied on the protile Recycle bin button Deletes all drawing elements or the one just selected Line width button Change of the line width of the drawing elements Color button Clicking on the Color button opens a color selection box where the color of the drawing element can be selected with a click of the mouse x1 button Resets the zoom factor of the protile diagram to Its original size B 45 0019 e 4 297 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 2 z Histo measurement mode in 2D display e Click on the Diagram button in the Measure button bar Cl
409. on of a macro the Stop Macros window is always displayed on the screen This enables a macro to be stopped any time by pressing the Stop button The functions of the sample macros are explained below 4 8 4 1 Distance Macro This macro permits measurement of the distance of a line created in the scan image e Click on the Distance button in the Macro subordinate toolbar An XY scanning image of the specimen is recorded and displayed At the same time the Mouse position test window appears on the screen e Then draw a line over the distance to be measured by clicking and holding down the mouse button The click of the mouse sets the starting point releasing the mouse sets the end point of the line After release of the mouse button the length of the line in the scanning image is displayed in um Any required number of lines can be defined in the image The previous line is deleted e A click on the Exit button in the Mouse position test window will end the macro 4 8 4 2 Profile Macro This macro permits the gray values of a line created in the scanning image to be determined pixel by pixel e Click on the Profile button in the Macro subordinate toolbar An XY scanning image of the specimen Is recorded and displayed The Profile window is shown on the screen e Then click and hold down the mouse button to draw a line in the scanning image for which the gray values shall be determined The current numbers of t
410. ons Create type Database Files nidb Cancel Fig 4 19 Save Image and Parameter As window and Create New Database window 4 30 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan amp Projections mdb AIM OPERATION File Menu Recordset 0 o0 Name Description Notes f User m Acquisition Date Time Scan Mode Scaling Position Pixel Time Objective Beam Splitters Wavelength Pinhole Stack Size Save Image and Parameter As Name Conv 7 Description Channel Fluorescence Notes User Jappcenter Database MDB SRE pea BE ek nett hl Geta C image Data Database_Rel 3_O0 Lambda_Stack mdb Lambda_Stack mdb C lmage Data Database_Rel 3_O0 Test Rel 3_0 mdb T est Rel 3_0 mdb C lmage Data T ag der offenen Tuers Demo Bilder mdb test mdb test mdb C lmage Data T ag der offenen Tuer Demo Bilder mdb Demo Bilder mdb Compress Files M Total 252 000 kByte Fig 4 20 e Click on the OK button in the Save Image and Parameter As window The Projections mdb AIM window now shows the saved image Selected 0 Bytes Current 0 Bytes Database window Carl Zeiss The Recordset box indicates the current number of the image in the image series contained in this database e In the Description text box you can enter for example the configuration of the image e In the Notes text box you can ente
411. operation for at least one hour 3 Calibration procedure e Click on the Scanner button in the Maintain subordinate toolbar of the Main menu This opens the Scanner Calibration window e Click on the Speed 1 10 electr or Soeed 11 13 electr button respectively e Activate the Display Graphics check box enables to check the progress of the calibration process on the Progress Status bar During successful calibration process the status button is of green color in case of an error it switches to red The progress of the calibration process is indicated by the Progress Status bar The calibration process is completed when the indicator button is grayed e Click on the Calibrate button to start the automatic scanner calibration e Confirm warning information with OK e Click on the Close button to close the Scanner calibration window The More function is for servicing purposes only and can only be performed by authorized personnel Its access is therefore password protected 4 10 1 2 Important Notes and Hints The tuning procedure runs automatically to a large extent without any problems However several errors can occur That s why it is strongly recommended to observe the complete calibration process If a status error message occurs or the calibration procedure is not finished properly this can have the following reasons Non regularities of the scanner feedback L The ticks on the outer sides of the grid vary about m
412. or coded height slices Each level i e each slice is assigned a different color For direct evaluation a color scale is shown indicating the actual height above the bottom slice Orthogonal sections This computation produces a triplet of mutually perpendicular sectional images Oblique sections A section through the stack is made along an oblique plane defined by the selection of five coordinates i e X Y Z angle of rotation and angle of tilt Topography option A computing program for surface topography presentations as required in materials research is available Physiology option With a special software kinetic processes can be tracked which is especially of interest to physiology Image VisArt option Three dimensional display of floating transparent structures cells or opaque structures material 3D Deconvolution option 03 06 B 45 0019 e 3 3 INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 LIVE Carl Zeiss Optical Diagram of the LSM 5 LIVE LSM 5 LIVE DuoScan 3 3 Optical Diagram of the LSM 5 LIVE Schematic Beam Shaper shutter Ocular Aperture Tube Lens Scanner Optics Scan AchroGate Confocal Optics Aperture d x y CCD Line 2 Confocal Aperture d x y Diode Laser 635 Diode Laser 532 Diode Laser 488 Diode Laser 405 CCD Line 1 Microscope Scan Module Laser Module LIVE AOTF Acousto Optical Tunable Filter Fig 3 2 Optical path schematic 2 channel configuration The d
413. or each measurement The object features generate a result value for every single object 6 52 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss The dialog shows two lists One shows the Available Features as groups on the left The other one shows the Selected Features Double clicking on items of the left list will add the Selected Features to the right list Double clicking on an item of the right list will remove this item trom the list Selected Features can also be transferred by clicking on the button in the middle lt lt gt gt of the dialog The combo box above the right list represents predetined feature lists Selecting one of the entries will Till the right list with these features previously selected features will be overwritten The button Select All will copy all features to the list of selected features The button Remove All will clear the list of selected features Clicking on the Apply button will execute the measurement process and switch to the General tab sheet of the dialog Parameters Available Features List of available object features Selected Features List of selected object features Select All Select all available object features for measurement Remove All Remove all object features trom the selected features list The following sections describe all measurement features which are defined in the system 03 06 B 45 0019 e 6 53 Carl Zeiss Object Features geomet
414. or image display Use of this function is recommended to find the real surface in the case of images with pronounced noise All image pixels with intensity less or higher than the thresholds set are ignored for the surface calculation Intensity Threshold ES e Click on the Intensity button to select the intensity thresholds for the surface generation Lower threshold se The Intensity Threshold window appears Upper threshold P e Set the lower and upper intensity thresholds using the appropriate sliders Fig 4 281 Intensity Threshold window e Click on Close to close the Intensity Threshold window 4 280 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Height threshold Z Threshold x Click on the Height button to calculate the 704000000 00000000000010000000071100011009000COIKCO0IO OOOO AEO upper height thresholds for image display Use of this function is recommended to get rid of unwanted peaks and valleys taken into account for Fig 4 282 Z Threshold window parameter calculation All topographic data with height values less or higher than the thresholds set are ignored for the display and parameter calculation This threshold applies both for 2D as well as for 3D topography display modes Upper threshold e Click on the Height button to select the intensity thresholds for the surface generation The Z Threshold window appears e Set the lowe
415. or the other slices 4 134 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss 4 6 9 4 Preview Panel erences otart The Preview function enables you to see the result Preview Slice H i eez of interpolation for one slice each in a preview Erd window Fig 4 127 Preview panel e Activate the W Preview check box with a click of the mouse The Interpolate C amp B Preview Image Display window will be displayed At the same time the Slice slider with the relevant input box and arrow keys and the two buttons Start and End are displayed in the Preview panel e Use the slider or input box arrow keys to set the slice which shall be displayed in the preview window e Clicking on the Start or End button permits the fast activation of the Start Image or End Image for previewing e Deactivate the Preview check box to close the Interpolate C amp B Preview Image Display window 4 6 10 Channel Shift The Channel Shift function is used to produce a congruent image with relation to the pixels of the various channels i j aii AM HeX Y a gt ae gt L Ar Unnamedg2 This pixel correction function is particularly important in UV applications A Horizontal oo fo Vertical a fo 5 Channels f Chi Ch3 e Click on the Shift button in the Process T Preview subordinate toolbar of the Main menu This opens the Channel Shift window Fig 4 128 Channel Shift wi
416. or the scan info in the Font menu a different type font and type style 4 13 25 2 Context Menu for Topography Images When transferring a topography to the print preview you can change the size and shape of type and scale lines for the 3D graphics and protile measurement results 1 Context menu for 3D graphics e Click on the right mouse button A context menu with the options Font enlargement and Line width enlargement is displayed e You can change the type size in the Font enlargement menu and the line width of the scales in the Line width enlargement menu 2 Context menu for Profile measurement function e Click on the right mouse button A context menu with the options Scaling font enlargement Marker font enlargement and Overlay font enlargement is displayed e You can change the type font in the Scaling font enlargement menu the size of the marker table in the Marker font enlargement menu and the type size of the red measurement results in the Overlay font enlargement Print Setup 4 13 25 3 Arranging and Printing the Print Printer Preview Hame KODAK sL5 S600 PS 2014 105 Properties Status Ready e Click on the Arrange button for optimum l a e a A layout of image size and position relative to the o textual information Paper r Drientatior e A layout generated with Prev Preview can be Size Letter Potrat printed by clicking on the Print button in the Print toolbar Source Automatically Select
417. ore than 1 tick width between consecutive images in the middle of the calibration process the linearity is optimized and the problem starts to occur e Stop the calibration procedure e Call the LSM service hotline If optical calibration comes not to a successful end please contact your service hotline 03 06 B 45 0019 e 4 211 OPERATION Maintain Menu Carl Zeiss 74 Objective Control Objective Focus Speed Partocal Correction Objective Position Mame 1 Plan Meofluar 1080 3 Plan Neofluar 20 05 Plan Neotluar 40 0 75 Plan Apochromat 631 4 Oil Empty position Empty position Fig 4 220 Objective Control window Change Objective a Favorite Objectives es B Empty position bia B C Apochromat 1040 45 W Plan Apochromat 63 1 4 Oil Close ve B Plan Neofluar 105 0 3 Remove Plan Neofluar 25x08 Imm cor Phe Objective z B Plan Meofluar 4071 3 Dil a User Defined Objectives Add Bnew objective Objective Eg Potential Objectives Edit Objective Fig 4 221 Change Objective window LSM 5 LIVE LSM 5 LIVE DuoScan 4 10 2 Objective This function permits changed objectives to be activated and the parfocality to be set without having to exit the software 4 10 2 1 Objective Change e Change the required objective in the nosepiece e Click on the Objective button in the Maintain subordinate toolbar of the Main menu The Objective Control window appears on the screen
418. orm mode allows only one image to be copied or moved The Gallery and Table modes permit several images to be copied or moved simultaneously by multiple selection keeping the Shift key pressed on clicking e Open the relevant databases and position both windows side to side e Select the required images multiple selection by keeping the Shift key pressed from one database e Click on a selected image and keep the mouse button pressed move the mouse button to the window of the other database a small rectangular appears near the mouse button and release the mouse button again Drag amp Drop 03 06 B 45 0019 e 4 25 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan The images are now being moved to the other image database i e they are deleted from the first image database and are then only available in the second image database e f the images shall only be copied also press the Ctrl key during the Drag amp Drop procedure in addition to the rectangular a sign will also appear near the mouse button The images will then be available in both image databases lt q 4 4 3 8 Display Images with Certain a Features Filter aes The Filter function permits the display of the freemen M Bot a database to be modified in such a way that only images with certain features are displayed This requires the definition and following activation of a relevant filter Defined filters can be stored reloaded an
419. oscope Use of the optional piezo objective focusing device The HRZ Step slider is used to set the step width of the fine focusing device e Use the arrows A and W of HRZ to move the Tine focusing device upwards or downwards in steps As soon as the focus position is changed via handwheel or software the piezo objective focus is automatically leveled e A click on the L button moves the piezo objective focus in the center position of its travel range and the focus position is reset accordingly Therefore the same Z level remains visible the current position is not set to zero The motor focus of the stand is operated in the same way via the relevant buttons Moving into the Work or Load position is always performed via the motor focus and not via the piezo objective focus LS Please see CHAPTER ANNEX of the printed manual for further information on the piezo objective focus 03 06 B 45 0019 e 4 115 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Stage Position 4 5 9 3 Stage Position Panel The Stage Position panel shows a symbolic specimen carrier in the left upper The buttons for moving to a position and mark it To 3 1 2 ee are below or on its right ev step pm o Curent Pos x 1988 00 y 1988 00 Marks gero q OOO y 000 z OOOO Mark Pos Move To Remove Remove al HAZ ero The scanning stage can be moved using the Fig 4 103 Stage Position panel joystick or
420. oscope Configurations list box e Click on the Apply button e Click on the Close button to close the microscope window f Only those microscope settings which are encoded and motorized can be reloaded Store a microscope configuration A newly created or changed microscope configuration can be stored under a new name as follows e Enter the desired name in the first line of the microscope setting list box e Click on the Store button e The actual configuration with the chosen name is added to the microscope settings list e Click on the Close button to close the microscope window 03 06 B 45 0019 e 4 47 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Delete a microscope configuration A no longer required microscope configuration can be deleted as follows e Select the microscope configuration to be deleted from the microscope configuration list box e Click on the Delete button e Click on the Close button to close the microscope window Assignment of a microscope configuration to a button A microscope configuration can be assigned to a button as follows e Click on the Assign button e This opens the Assign Microscope Settings To Button window e Click on the arrow in the Button list box and select a button out of the list ci With increasing numbers the buttons are arranged from the upper to the lower row from left hand side to right hand side e Click on the arrow in the Settings list box and
421. ottom of the Macro window match the currently used objective e Click the button Calibrate Meaning of the Graph Figure ARUP E on Brown Focal plane of the CCD Line Detector tt iS LAWL Red Focal plane of the collimator Pie artes Orange Estimated focal plane pe The brown and red graphs should be close to this curve Fig 4 181 Graph of Focus Function 4 182 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss e In order to follow the calibration process just simply click into the Message window and a scroll bar will appear Fig 4 182 e The Calibration Matrix takes about 25 min for each objective e After the macro has finished a message will pop up with a the request to switch the objective lens in order to make the changes effective e f necessary repeat the procedure for the other objectives e Use the Update Diagrams button in order to check if an objective has already been cali brated Select an objective press the Update EEA E Diagrams button Fig 4 182 Collimator Calibration window after If no graphs appear this particular objective calibration has yet needs to be calibrated e Click Exit to finish Meaning of the Graph Figure Each coloured line represents an individual laser line Each coloured calibration point represents an emission filter Standard graphs are displayed with black points
422. oughness measurement mode in 2D display Profile display e Click on the Profile button in the Measure button bar e Click on the Roughn button in the Measure button bar The roughness parameters are calculated and displayed on the left below the image All roughness parameters calculated from a 2D profile are named with R The Copy button is displayed below the right hand side of the image This button permits the roughness parameters to be copied to the clipboard and imported to another program e g MS Word or MS Excel via the Paste function 2D Amplitude parameters Profile Roughness O i eme e e w Root mean sauare deviaton ta Pa wa em 03 06 B 45 0019 e 4 299 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan Calculation of roughness parameters The following roughness parameters are calculated e g for a Y section Mean height of all profile height values R z y a x y Nx Ny number of pixels in X or Y direction J at N3 I Arithmetic mean deviation of all profile height values R y clay J R Skewness of the distribution of all profile height values Rsk Loon R gt oz a NR Se y Kurtosis of the distribution of all profile height values Rxy i 7 4 Rey gt ez x y KU N R 2 V Maximum peak height Rp Rp Zima R max C Maximum valley depth Ry Ry R 7 Z min Max
423. out regard to the overlay element If of importance the length and perimeter of a line figure the area of a closed figure and the inclination angle of a single line will be displayed On deactivation of the Measure button the measuring value of the selected element is no longer displayed and all the elements created afterwards will not be assigned with a measuring value B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss Extract Region E 03 06 Off button Deactivation of the display of overlay elements in the Image Display window hide overlay Deactivation of the overlay functions Line button This button allows you to determine the line thickness of the area outline Cut button The image contents of an overlay element are cut out and the area will then appear in black Copy button The image contents of a closed overlay element are copied to the clipboard Paste button The image contents of an overlay element copied to the clipboard are inserted in the current Image Display window and can be positioned anywhere in the image using the mouse Undo button The last Cut or Paste action can be undone by clicking on the Undo button Extract Region button The region of a Z Stack or 4D image surrounded by an Overlay element is extracted and can be displayed and stored separately in a new Image Display window This function is only active if an Overlay
424. pboard activate the check box Clipboard 6 58 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss A single object of interest can be visualized Clicking on a specific row in the data grid chooses the object By selecting a row in the data grid a new image is created with the object of interest visualized The visualization depends on the settings in the Object Visualisation field If Render is chosen the object of interest is displayed with the Surface Rendering method If Mask is chosen the object is labelled in a pseudo colour in a new image stack Parameters Mask Image Dens Image Object Volume Label Clipboard 03 06 Single channel mask image sequence that defines the objects Image sequence for densitometric measurement and property source Stores measurement values of objects including database filename Stores volume measurement values of objects including database filename Generates an image sequence with all objects labelled in different pseudo colours Measurement values are automatically written to the clipboard B 45 0019 e 6 59 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 7 ANNEX Contents Carl Zeiss ANNEX CONTENTS Page 7 PENS Nessa tateenes cn te Sam E E OA 7 2 7 1 Recommendations for Excitation Laser Lines and Emission Filters Of Dy S ccccccceeeeees 7 2 7 2 OW Ue ROMS OV CROW ysna O ees Selena 7 3 vey ie LSM 5 LIVE 1 Channel Biomedical CONTIQUIATIONS
425. pe Shape If Input is a multichannel sequence any number and combination of channels can be selected Output will only get the selected channels as results The Count scroll bar determines the number of recursive operations The following shapes numbered 1 to 3 from left to right are available amp If Grey Morphology is selected the function will respect all grey value shades of the sequence Input If Grey Morphology is not selected the function will distinguish between O and non O only The result Output will be a binary sequence Parameters Input Input image sequence Output Resulting image sequence Shape Shape used 1 cross 2 cube 3 cube cross Count Number of recursive operations 6 32 Grey Morphology 0 Distinguish between 0 and non O only 1 All grey value shades are taken into account B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Segment Interactive This function carries out a grey value segmentation by means of thresholding Interactive Automatic Input ho Output 2 Hew Colour Green C Blue Red Binary m Invert i Le a fiss al 4 oss ao 255 OK Cancel Input Histograrn Fig 6 24 The Interactive tab sheet of Segment dialog window must be selected Segmentation is especially used to generate binary regions These are required for the measurement Two threshold values determine which grey value range of the Input imag
426. pecimen within the limits determined in Num Slices and Interval the cutline is determined via the Line Sel function B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 4 2 Frame When the Frame button is activated a frame of variable size is scanned pixel by pixel and line by line The laser beam is moved over the specimen line by line The scan parameters and the channels single detector are set via the Mode and Channels buttons and the laser settings can be checked again or changed 1 Mode When the Mode button is activated the Objective Lens Image Size amp Line Step Factor Pixel Depth Scan Direction amp Scan Average and Zoom Rotation amp Offset panels are displayed in the Scan Control window Objective Lens Image Size amp Line Step Factor panel e Open the Objective list box and select the objective to be used via a click of the mouse identical to Microscope Control When using immersion oil objectives make sure to perform immersion as required e Select the Frame Size from the default sizes via the buttons 128 256 512 768 1024 or enter the required values via the keyboard Recommended setting to start with 512 x 512 pixels Itis also possible to enter different values for X and Y The value for Y is freely selectable between 1 and 1024 pixels integers The value for X depends on the chosen image format The most common image formats can be set directly with the Frame size bu
427. processing o on 4 279 4 281 Content of display WINdOW 6 13 Display MOdES cccceeeeeee 4 284 4 287 NAG Cs er a eee des 4 29 6 12 Measurement functions 0 cece 4 294 IMAGE SEQUENCE eee seereeeee teers teri 6 12 GUNG ie acme es cases outee aceon E ees eeene eet 4 51 SCANMG NMAC Cri aiai 4 145 Track ConfiguratiOn cccccccccececeeee R254 e523 SCA AV CNA 6 espeor ee Snae 4 321 Tracks panel list Of 4 61 SCail COMUO isare 4 68 Trigger interface LSM 5 LIVE cccccccccscescesceee 7 10 SCAN SPCC se ee sees 4 71 4 321 TUPI power off s es 4 326 Scatter CLAGK AIM eaha a raa 4 257 Segmentation U A E S A SANA n TT 4 137 Biel cre lcigiaccre Tte eee ee een ee eee 4 131 Select Object in specified size range s 6 41 V DS LMC E E E E TAAT 4 200 Sharpness filter erroni aasan 4 130 po ete ae Shut down procedure n onrnrnrnn 4 325 Mask grey value eao SINGIG CMAN Vln distecincciciatee EN 4 315 Scale a range of grey values u 6 23 SUNOS MAGI E EEA 4 51 Scale ON EY VaIUC aerie oE 6 21 SIIGIOPUACKING sa ri 4 61 Vertical scale of histogram s 6 20 STAGE WAC KING ixciecrtentasaustontutarentoateusasaanntcl 4 55 VOW Menesiai tad tice ertialaai pact Os hobantes 6 6 SE E PE en ee ee ae ee 4 229 6 2 Visual macro editor 4 177 4 189 Soles gree ee sen ee Cer ter 4 78 4 245 4 323 Volume features BAG eae ae eae 4 114 DeNsItOMEtHIC sees tet teeeiees 6 56 Starting the LSM 5 program osaan 4 13 C Guaeemerte ce e
428. r For regions voxels that are at the edge of the image sequence it assumed for erosion that there are white voxels with a grey value of 255 4095 outside the edge For dilation it is assumed that there are black voxels with the grey value O outside the image sequence If the Grey Morphology tickbox is activated erosion sets the grey value of the central voxel to the minimum of all neighboring voxels affected by the structural element dilation sets the grey value of the central voxel to the maximum If the Grey Morphology tickbox is not activated the neighboring voxels are only distinguished by grey value O and non O For erosion the central voxel is set to O if any of the neighbors is O It is set to 255 4095 if any neighbor is not O For dilation the central voxel is set to 255 4095 if any of the neighbors is not O It is set to O if all neighbors are O Erosion reduces the size of bright regions separates thin connections between them and makes small regions disappear Dilation on the other hand makes bright regions of the image grow in size fills gaps and smoothes small contour details 03 06 B 45 0019 e 6 27 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan The result of erosion and dilation is called opening On the one hand this maintains to some extent the Original size of the regions while not losing the smoothing effect of erosion on the image This name stands for the operation of reducing convex bu
429. r Coded Bi 16 0 aya s 0 eee eee eee eee eee oe 4 152 Open Close the Depth Coding WINKOW cccccccecccceecccceeeeceeeeeeeteeeeeeeseeeeeeaeeeeeeneeeeas 4 152 SETS Aa E EE ae nT ee en eee ee ee ee ee 4 153 BEE eaea i Peer eee E tr ere See eee a eran eee nee E reece E eer eee 4 153 eu aN Po eee ee ee ee ee EE 4 155 FO CT ON he ascetic ssa ET E E E ay oes geese EA cape EAE ow E 4 155 Open Close the Projection WINKOW cccccccceseececeee ee eeeeeeeeeeeeeeeeeeeeeeeeeseeeeaeeeeeeeaneeees 4 155 SONS saree teeth ose hata E EE E E E E E E EE E A 4 155 KEES A E E EE E OEE E E TAE E E E ET 4 156 Preview PAINE cncsnctovacesaacaneiaaroesdtetandsenssouaasshdduaeasaeiasadabsnadassadguaauaianaroraanniedaibeadamecipeaaeties 4 157 AO ae ects pts pre sco rsd seed ge tt speedos ne se we E E 4 159 Open Close the Stereo Images WINKOW ccccceccccceeeccceeseeeceeeseeeeeeeeseeeeeeeneeeeeeeaaeeeeees 4 159 OIC ANG E ourtine ope EA E E E EEE ET A E T EE 4 159 SUN WASP ANC E E EN E E A N EEE E ET 4 160 PROVO Pa an EEE EAEE AAE TAA AA E A 4 162 3D DeConVolution COC VW arescasenascasrcgenaectuanoiacaetacescesantaseresaraiccmenenud tet aeheustnasaameaneteatensseatae 4 163 Open Close the 3D Deconvolution VV IMG OW dic 25ctsttedn depdtncesieddan tind sxanciosdetdcaindeonx enantio 4 164 SOUN CE PANE cnsosasientecoe metas cacgaincspanasobsttaveadantaciaadaesasnsissanteaseieceadentedanstetactsatosaanetsaniriatanies 4 165 Deconvolution Panel srcee
430. r an XZ scan in fluorescence and is also only computed for this e Activate the 2D DCV button in the Orthogonal Sections toolbar If the Fast button is activated calculation of the 2D deconvolution inverse DCV mode is performed immediately The 2D DCV settings button can only be activated if a licence for the 3D DCV option has been purchased Otherwise this button is grayed e Click on the Settings button The 2D Deconvolution window is opened The 2D Deconvolution window contains the Deconvolution panel with the two tabs Method and PSF 1 Method tab Method PsF The Method tab enables you to choose between Method ince O the calculation methods Inverse and Iterative Restoration Effect Weak oo Strang For more details of explanation of deconvolution j Auto detect and the calculation methods see 3D DeConVolution page 4 163 In the Inverse method the Restoration Effect slider can be used to set the signal to noise ratio between Weak low noise and Strong pronounced noise Fig 4 256 Method tab Activation of the Auto detect check box starts a routine for the automatic determination of the noise level in the entire image part of the Z Stack If Auto detect is enabled the Restoration Effect slider is disabled The Iterative method permits in addition to the parameters of the Inverse method the maximum number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop function to be
431. r and upper intensity thresholds using the appropriate sliders e Click on Close to close the Z Threshold window Auto Z By clicking on the Auto Z button the surface topography is displayed in the Image Display window in that way that it is automatically normalized to the lowest and highest Z value of the 3D topography e Click on the Auto Z button The topography is automatically normalized with respect to the highest and lowest Z value 4 13 24 4 Processing by Filtering 1 Topography smoothing The three buttons in the Filter button bar allow activation deactivation of the filter functions for surface smoothing None button No filter for input data Median Gauss Aver Smoothing of z data using a low pass Median Gauss or average filter button Clicking on this button opens a selection box where the number of neighboring pixels to be used for filtering can be specified 1st row small smoothing via Median Gauss filter Median 3 x 3 5 x 5 7 x 7 2nd row medium smoothing via Average 9 x 9 11 x 11 15 x 15 3rd row pronounced smoothing via Average 25 x 25 35 x 35 45 x 45 03 06 B 45 0019 e 4 281 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan e To investigate the effects of various filter modes select one of the 3D display modes Profiles Grid Filled or Shaded from the Display button bar e Click on the Median sub button to set the smoothing of the integrated Median filter
432. r further information about the image content 03 06 B 45 0019 e 4 31 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan 4 4 5 Import of Images The Import function enables the import of externally created images into the Image Display window and the image database of the LSM 5 software e Click on the Import button in the File subordinate toolbar of the Main menu This opens the Import Images window Import Images El x Convallaria lem Image type Single Image I eA Look in E Images al d EET EE a conail1 8f lem Proj Connoil af lem cal FLO n usal z lem Convallaria lsrn a usal 3 lam Convallaria2 sm n ual 4 lam conyvcontocal lam a UY DIC lem convetack lem P12 2512 1 3 channels 146 23 pm 146 23 pm File name Eornvalaria lsm __Gren 823 703 kEpte z Files of type A Supported Files Cancel Fig 4 21 Import Images window e Select the data medium and the directory where the relevant image is contained in the Look in selection box e Select the image Tile by clicking on it The selected image will be shown for checking in the Image Display window on the left together with the relevant details size channels storage volume e Select the image type Single Image or Image Series in the Image type selection box e Click on Open The image is displayed in a new Image Display window All the usual image a
433. r range To delete a defined color click on the relevant color button and then on the Remove button Standard colors black for OFF red green blue and white cannot be removed e Click on the Close button to close the Channel Colors window Newly defined colors are accepted and displayed in the Channel Color Selection window They can then be used in the same way as standard colors The PMT1 photomultiplier is activated deactivated by the check box e Proceed in the same way for the other PMT photomultiplier 4 58 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 5 Beam path Laser attenuation Excitation e On the Beam Path and Channel Assignment Line active Transmission B t panel click on the Excitation button C sonm 01 gt A This opens a dialog box of all available lasers C 488nm 01 4 ID with their wavelengths and their usable C 532mm a1 gt attenuation Bo 635 nm ua AA gt e To select the desired laser line activate the check box for Line Active Xe amp amp e Use the Transmission slider to set the utilizable laser intensity recommendation start at 50 The transmittance of the attenuator changes accordingly Fig 4 42 Configuration Control window e This allows you to adapt the laser intensity very sensitively to the job Activate the check box for Line Active rs By clicking on the Excita
434. r x This function permits new filter sets to be added to Piir Cubes Stand Daecriptian oom the database Producer Carl Zeiss Jena GmbH C Non Zeiss re Name pply For this proceed as follows Series Number e Click on the Add New button on the Edit sa ae Filter Beam Splitter List window E 5 The Add New Filter Beam Splitter pices E window is opened G ee E Tr e Enter the data of the new filter set in the Filter ie Cubes Stand Description panel then click on Naw RerGubes Sind the Apply button The new filter set is stored in the database and included in the New Filter Cubes Stand panel You can now activate the filter for a filter wheel position using the procedure described above gt Ifyou have activated the Non Zeiss check Fig 5 2 Edit Filter Beam Splitter List box filter sets from other manufacturers window can also be included in the database e To remove an new filter set from the database select it with a click of the mouse in the New Filter Cubes Stand panel and then click on Remove e Click on Close to close the Add New Filter Beam Splitter window e Click on Close to close the Edit Filter Beam Splitter List window e Click on the Store button to accept the new settings e Click on the Close button to close the Emission Filter amp Beam Splitter Control window When you start the LSM 5 LIVE software the filter data are updated 03 06 B 45 0019 e 5 3 TOOLS LSM 5 LIVE Carl Zeiss Stand Select LSM 5
435. rack is not available as a single track configuration but only within the recording configuration To edit a track name within Recording Configurations proceed as follows e To select the track click on the relevant track name in the List of Tracks panel Then click on the name again to open the text editing field e Change the track name via the keyboard Use Esc to undo the procedure e Click once in the area outside the text editing box to close this box 3 The channels of the individual tracks with the relevant scan parameters can be displayed in the Scan Control window after activation of the Channels button The description of channel 1 in Track 1 for example is ChL1 T1 3 Config button in Multi Track mode The Config button in the Multi Track mode permits all tracks to be loaded stored under any name or deleted a Load a Channel Mode Configuration An existing recording configuration can be loaded as follows T Channel Mode Configurations e Click on the Config button the Channel Stare Apply Configuration F Mode Configurations window appears on the 02E Configurations a p screen Store Apply Delete e On the Store Apply Configuration panel click on the arrow button ZA Fig 4 45 Channel Mode Configurations window This opens a list box of all stored recording configurations e Browse through the configurations by clicking or use the scroll bar at the side of the list box 4 6
436. ral a space of 1 8 m x 3 6 m for systems with a 0 or 45 oriented computer workplace or 2 8 m x 2 6 m for systems with a 90 oriented computer workplace is required As mentioned free access to the long side of the electronic and laser system rack is required for service and setup of the system If possible we also recommend to have a free space of 30 cm to the next wall all around the system 2 5 Dimensions Overview Component Overall dimensions Science desk microscope table wide 125 cm x 100 cm Side port systems Science desk microscope table narrow 100 cm x 125 cm Side and Rear port systems Electronic and laser system rack 75 cmx 115 cm Long side must be accessible Computer workplace 0 or 90 120 cm x 80 cm Computer workplace diagonal 45 130 cm x 160 cm Recommended for ergonomics Complete system in ideal configuration wide 180 cm x 360 cm table 45 computer workplace Complete system in ideal configuration 180 cm x 340 cm Most often used setup narrow table 45 computer workplace Complete system square setup 90 computer 280 cm x 260 cm workplace orientation i 03 06 B 45 0019 e 2 UJ Carl Zeiss 2 6 2 7 2 4 LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE Power Requirements LSM 5 LIVE DuoScan Schematic Outline of a System Service and Setup Access Fig 2 1 Schematic outline of a system Space Requirements Power Plug m Fig 2 2 LSM 5 LIVE with microscope table
437. rameter with size reduction from image database to Image Display window Reuse scan parameters of the selected image without loading the Image Refresh Image Database window Copy selected images to clipboard Paste images from clipboard into image database Select or edit search filter from image in the image database Switch search filter on or off Delete selected images from the image database The status line which displays the following current parameters is at the bottom of the Image Database window Total Display of storage volume of the entire image database Selected Display of storage volume of the selected image s Current Display of storage volume of the current image Clipboard Display of storage volume of the image s contained in the clipboard 4 20 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan File Menu Carl Zeiss 4 4 3 1 Form Display Mode When a image database is opened the Form display mode is used if no other settings were made under Settings in the Options subordinate toolbar E Convallana mdb Al Record a af 5 Name Jfigure2 Description Motes User hpame Acquisition Date Time Tuesday 01 04 2005 03 32 39 PM Scan Mode Plane original data 8 bt Stack Size 256x 25681 920 9 pm 920 9 pms DD pm Scaling 3 6 pm s 3 6 pms 1 0 pm Position we D O pm y D0 pm z 00pm Pixel Time 3 20 ps 000 Objective Plan Meofluar 10
438. rder to obtain an image of the selected object plane as a whole it is necessary to scan the object plane in a point by point line by line raster by means of an XY light deflection system The detectors as a rule photomultipliers convert the optical information into electric signals This allows the image of any object plane to be generated and stored within less than a second By a defined focusing Z axis movement it is possible to look at any object plane of interest By scanning a succession of object planes in a specimen a stack of slice Images can be produced Fig 3 1 Principle of confocal imaging This way the LSM technique in conjunction with ICS optics Infinity Color Corrected System has brought decisive improvements over conventional microscopy in terms of resolving power and confocal depth contrast Object features in the order of 0 2 um can be resolved and height differences of less than 0 1 um made visible without the use of interference methods 3 2 B 45 0019 e 03 06 LSM 5 LIVE INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 LIVE Duo Scan Three Dimensional Presentation of LSM Image Stacks Carl Zeiss 3 2 Three Dimensional Presentations of LSM Image Stacks One of the advantages of the LSM technique is that it can present structures in three dimensions This opens up many ways to process images Outlined below are some of the possible methods to extract Spatial information from stacks of slice images Gallery
439. re valid for All or just for one Defining the opacity for the channels independently is useful when the brightness and contrast of the channels differ too much Threshold defines the range with no opacity It is completely transparent The range starts at grey value 0 The length of slope is detined by Ramp The maximum opacity value is set with the parameter Max Opacity This range ends at the maximum grey value The Opacity Table shows the grey value histogram of Input with the opacity definition as a red line When Auto Update is selected the reconstruction is updated automatically whenever a parameter is moditied except Input Output or Number of Views Show Cube defines whether a wire frame cube is shown in the Display window or not Application 1 This method can be applied if the structures in the Input sequence are unsharp so that objects are poorly defined by their grey value 2 In this case the Opacity Table is defined as a ramp Low grey values have weight O to suppress the background voxels The opacity rises with increasing grey values depending on the parameter Ramp The value of Max Opacity defines the weight of the high grey values High grey values above a threshold have weight 255 to show the object voxels unsuppressed Of course a smooth step can be used 3 The result is a display with inside structures shining through A 3D impression can be obtained by rendering with several view directions 4 In contrast t
440. rea sum of all triangles formed by adjacent data points of the surface reconstruction to the 2D surface area produced by projecting the 3D surface onto the threshold plane absolute flat surface gt is equal to base plane Sdr 0 The increase by which the 3D surface is larger than the basic plane e g 625 is a 3D surface which is about 6 25 times larger than the projected basic plane 03 06 B 45 0019 e 4 305 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 2 Profile measurement mode in 3D display This function is performed in the same way as in the 2D display mode with the following exceptions The buttons and I are replaced with the buttons 4 and Furthermore the Position slider and the input box information of the position of the intersection line in pixels are displayed below the Table and Profile button bar Changing the Z Threshold also results in a change in the profile In the 3D image a red marker line shows the y and x position of the displayed protile diagram 50 Delence burl Peni 902 x S129 15 3 charis Bly Fig 4 299 Image Display window Topography display 3D Profile e The position of the marker line profile intersection line can be changed by moving the Position slider IN X Or y e Press the x or y button to select the required intersection plane 3 z Histo measurement mode in 3D display This function is performed in the same way as in the
441. reation of an ellipse in the Image Display window The first click sets the center point the displayed line permits the determination of the first dimension the second click sets the first dimension the second dimension and rotation direction can then be determined the third click sets the second dimension and direction and ends the procedure Closed free shape curve button Creation of a closed Bezier figure in the Image Display window The first click sets the starting point each additional click adds a further line a click with the right mouse button closes the figure and ends the procedure SN Open free shape curve button Creation of an open Bezier figure in the Image Display window The first click sets the starting point each additional click adds a further line a click with the right mouse button closes the figure and ends the procedure Ol Circle button Creation of a circle in the Image Display window Clicking and holding down the mouse button sets the center point drag the diameter and release the mouse button to end the procedure gt Line with arrow button Creation of a line with arrow in the Image Display window Click and hold down the mouse button drag the line in any required direction release the mouse button to end the procedure A A Text button Creation of a text box in the Image Display window After clicking on A the Text window will be displayed and text can be entered via the keyboard The
442. rge highlights Large values generate small highlights similar to a spot Use the parameter Reflection to control the ratio of diffuse and reflective brightness components i e the overall basic brightness compared with the highlights When the value of Reflection is low the highlights predominate when the values are high the region appears to be uniformly illuminated and the highlights are not so pronounced When Auto Update is selected the reconstruction is updated automatically whenever a parameter is modified except Input Output or Number of Views Show Cube defines whether a wire frame cube is shown in the Display window or not Application This method can be applied if the structures in the Input sequence can be segmented by grey value thresholding Because the gradient is calculated for every pixel the Output appears in very fine detail Noisy Input sequences must be smoothed function Smooth before rendering otherwise the surface appears rough Parameters Input Input image sequence Output Resulting image sequence Number of Views Number of reconstructions to be calculated Angle X Angle of rotation on the X axis start position Angle Y Angle of rotation on the Y axis start position Angle Z Angle of rotation on the Z axis start position Channel All The following parameters are valid for all channels X The following parameters are valid for the selected channel only Grey Low Low grey value threshold of the regio
443. ric 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan If Object Features are selected one set of measurement data is calculated for each object Group Name Volume Volume Filled Ellipsoid Ellipsoid filled Surface Area Surface Area Filled Sphere Diameter Sphere Form Factor Number of Holes 6 54 Name Volume VolumeF EllipseA EllipseB EllipseC EllipseAF EllipseBF EllipseCF SurfArea SurfAreaF Dsphere Fsphere Nparts Description Volume of the object Volume of the filled object Length of the main axis of the ellipsoid with the same geometrical moment of inertia as the object Length of the middle axis of the ellipsoid with the same geometrical moment of inertia as the object Length of the minor axis of the ellipsoid with the same geometrical moment of inertia as the object Length of the main axis of the ellipse with the same geometric moment of inertia as the filled object Length of the middle axis of the ellipse with the same geometric moment of inertia as the filled object Length of the minor axis of the ellipse with the same geometric moment of inertia as the filled object Surface area of the object Surface area of the filled object Diameter of the sohere with the same volume V 6 VOLUMEF x Form factor of the object 5 Jz NOLUMEF SURFAREAF gt Number of holes within an object B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoSca
444. rigger Trigger out Trigger Fig 4 83 Stop Series panel e Open the Trigger in list box with a click on the arrow button e Choose one of the trigger keys 1 to 4 e g Trigger2 It is also possible to trigger an out signal via Trigger Control e Open the Trigger out list box with a click on the arrow button e Choose one of the trigger keys 1 to 4 e g Trigger2 In this example the Time Series is ended on pressing key 2 of the Trigger Control and an out signal is given at the same time 03 06 B 45 0019 e 4 97 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan rs Ifthe entered number of cycles has been processed without a trigger impulse having been given to end the procedure the Time Series is finished If a trigger signal is given before the cycles have been processed the Time Series will only be interrupted Waiting for Trigger will be displayed in the status line The Time Series can now be continued via a new trigger signal or ended via Stop 2 Stop via Time Manual Trigger Time To end the Time Series via the time set on the PC Time button activated the end time must first be Humber b i SAL f entered in the Time input box Time hh 11 mmf 10 z5 joo Trigger out Mone Fig 4 84 Stop Series panel e Enter the end time via the keyboard Then click outside the input box once to close the box e Click on the Time input box to open it L The Time Series is interrupted w
445. rogram Call ea Se Uma steht tal oeiet cadid cedigaen ena a aa eehon acess 4 207 4 9 2 15 EEE E A ae nen a AE ne ORO re eae E See EE 4 207 4 9 2 16 MAO CHD ISDIAY ease ee a EAA EE Sa 4 207 4 9 2 17 e AIEA E EEEE EE A A EE EA EE A E A AEE AET EEA 4 208 4 10 Maintain Men assein an aae EEEa 4 210 4 10 1 SCANNET aeeoea O N E Ee E O E maid sec aals 4 210 4 10 1 1 Heceli aO Naa a A A S 4 210 4 10 1 2 APO Ma Notes and HINS ainese n E A iin Bebaegnenepedabent 4 211 4 10 2 E EAE EI EAEE E FO ae Be REE Ree EPSE ET E ATE ETT 4 212 4 10 2 1 ODEA CRN E taisa a ters 4 212 4 10 2 2 FOCUS Ped CAN en a a tamales 4 214 4 10 2 3 KA E NAE E E o EE T TAE E E NONE feet E TA 4 214 4 10 3 POLES CUUSTIMEN asine E E A 4 215 4 10 3 1 Open Close the Pinhole and Collimator WiINdOW ssssssisssinesrinesrrerrrrsrrresrrrenrree 4 216 4 10 3 2 PROE ESN eaa E E AE E O ON Te 4 216 4 10 3 3 Cona O A a A aaa eee nade eee 4 217 4 10 3 4 Aperture and colimator AdiustMen Tirrnassrcneroneu sne aai 4 218 4 10 4 E EE E E ana T seeaueees 4 220 4 10 5 OOOO enstinn a a a iat a ts taht Soe ahah 4 220 4 10 6 PNA A aee E E N E ato aaa ae cacdoaemy aeons E E aemeanea meats 4 220 4 10 7 A go ERA ar P E SEE E E RE EAEE A T E EE ET E 4 220 4 11 WV ING OWE Men Oiciccesiteesnictincenssays cusnansnceiiecaiecnas5evnauiasae E 4 221 4 11 1 ROEE E E EETA E PEE casein uote E AET E A A A 4 221 03 06 B 45 0019 e 4 5 Carl Zeiss 4 11 2 4 11 3 4 12 4 12 1 4 12 2 4 12 3 4 13 4 13
446. rom CT Using Gray Level Gradients IEEE Transactions on Medical Imaging 5 1986 45 47 5 D Gordon R A Reynolds Image Space Shading of 3 Dimensional Objects CVGIP 29 1985 361 376 03 06 B 45 0019 e 6 49 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan 6 3 5 Functions in the Measurement Menu Measurement Concept Measurement is based on regions objects in three dimensional space Segmenting an image sequence generates these The image segmentation process produces a mask image that defines the region A region is a group of voxels that touch at the surfaces or at the edges but not at the corners 18 voxel neighborhood This is illustrated by the following example The voxels marked black in sequential image Z 1 Z Z 1 all belong to the same region as the grey central voxel in sequential image Z The volume view shows the neighborhood interrelationships as a 3D projection Sequential image Volume view 6 50 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Measurement Process The measurement process consists of three steps region definition checking of the validity of the regions and feature calculation Region definition Automatically trom the mask image Region validation check depends on Minimum volume Measurement condition Feature calculation depends on Shape of the region Densitometric value distribution of the region Feature parameters Valid
447. rom four formulas Type 1 to 4 for ratio calculation The relevant decimal values can be entered in the input boxes via the keyboard The entered values remain unchanged even after switchover to another formula and can be reactivated any time The formula type activated last is always used for ratio formation during the scan procedure If the input box does not contain any value at all or no Suitable value the useful value last used will be activated The ratio channels are displayed in the Image Display window see Fig 4 61 e Select the required formula and enter the relevant values Letters can be entered into the formula fields which will be valued as 1 it Is also possible to make no entry which will also be valued as 1 but will not be displayed Set by min max in Scan Control window Channels mode allows the definition of the display scaling according to the expected minimal and maximal values Excitation panel e In the Excitation panel you can select other lasers and vary laser intensities in the same way as in the Laser Control or Configuration Control window All parameters under Channels can be varied online OPERATION Acquire Menu Carl Zeiss 74 Scan Control ca ee Faa Channel Settings Choreis E EEE go Find Fast ety Single Type 1 Type 2 Type 3 Type 4 Stop ChL1 T1 fi Min o oo78 oe Cont lst Image f Max igaaa First i Images W Set by minimas Calculates inten
448. rom the images prior to unmixing Advanced Linear Unmixing restarts the unmixing process again if negative values are generated The negative values are set to zero and ignored for the next unmixing calculation Ignore Overexposed Pixels and Ignore Underexposed Pixels will exclude this pixels tor calculation of the unmixed images 4 138 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss Multichannel Unmixing allows to apply the unmixing algorithm to a multichannel image up to 8 channels without the use of reference spectra The calculation of residuals and the subtraction of background based on a background spectrum are not available when this procedure is chosen It will not work for heavily overlapping signals Linear Unrieing Try to avoid saturation of fluorescence Signals in the stack to be unmixed This will generate a high signal in the residuals channel To get the highest quality unmixing results please define an extra background channel if possible I dupeca D ipet Oveteopoced Pra E Ipae LI rude org pc yj F words Fig 4 133 Multichannel unmixing 03 06 B 45 0019 e 4 139 Carl Zeiss 4 140 OPERATION Process Menu LSM 5 LIVE LSM 5 LIVE DuoScan Display p aj Split sy Ready Al2 e512 2 channels 12 bit Fig 4 134 Image Display window after unmixing B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu
449. rt 200 M with sensor ring and contact ring for laser safety Fig 1 10 Blind cap for closing any port equipped with an interlock device 1 21 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss User Interface LSM 5 LIVE DuoScan 1 14 2 Mounting and Dismounting the Scan Heads The scan heads are connected to the microscope via an integrated safety interlock They can be moved between two microscopes Make sure the system is shut off completely before starting the following procedure IS Be aware that the LSM 5 LIVE scan head weights up to 19 5 kg Moving the scan heads between Axiovert 200 M Axioskop 2FS MOT and Axio Imager Z1 Fig 1 11 Electronic connection port of the LSM 5 LIVE scan head e Loosen the three screws on LSM 5 LIVE or LSM DuoScan scan head Fig 1 12 1 or Fig 1 13 1 Fig 1 12 Port connection between LSM 5 LIVE Fig 1 13 Fastening screws of the scan and Axiovert 200 M head at the front of the tube on Axioskop 2 FS MoT and Axio Imager Z1 e Slowly pull the scan head away from the microscope port or the tube For mounting the scan head onto a microscope make sure the pins and the electronic connections of the safety interface match closely Fasten the three screws on the LSM 5 LIVE or LSM DuoScan scan head Fig 1 12 1 or Fig 1 13 1 1 22 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan User Interface Carl Zeiss e Use the three fastening screws Fig 1 14 1 and 2 to attach or de
450. s are available Manual button The time series is started manually with a click on the Start T or Start B button Trigger button The time series is started via a trigger signal from Trigger Control Time button The time series is started when the set time is reached The internal computer time applies Time input box Input of the time for the start of the time series Time button activated Trigger in listbox Selection of the trigger key 1 4 with which the start is to be triggered Trigger button activated Trigger out list box Selection of the trigger keys 1 4 for the out signal Pre Scan If this box is checked before starting the Time Series a continuous scan is started but no images are acquired until the button Go wi which then appears on the lower right hand side of the control window is clicked 03 06 B 45 0019 e 4 95 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 1 Start via Trigger Manual Trigger Time For the start via trigger control Trigger button E activated first determine the trigger key which is Tiggerin J Trager I Tigger out Trigger to trigger the start of the Time Series Fig 4 80 Start Series panel e Open the Trigger in list box with a click on the arrow button e Choose one of the trigger keys 1 to 4 e g Trigger1 It is also possible to trigger an out signal via trigger control e Open the Trigger out list box with a click on the arrow button e Choose one o
451. s to be activated in the Image Display Toolbars tab under Settings see Options menu It is also possible to display the scan parameter of an image Info button immediately when an Image Display window is opened The data to be displayed can be defined see Image Status Display tab under Settings in the Options menu The set of functions available at the Image Display window toolbars depends on the type of image shown The LSM 5 software handles the following formats frame single image and Z Stack of images frame time series time series of images and time series with Z Stack of images 4 224 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss line time series time series of lines and time series with Z Stack of lines point time series time series of points 4 13 2 Display Modes The following display modes are available for the different acquisition modes Display functions Cs a B B B a ae sete coded Cl oro O Mx w E Saey O m E o E E P __ E E E E e Mean Select so only active in case of multi channel images Inactive optional 03 06 B 45 0019 e 4 225 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan All display functions are exclusive functions Only one can be active at a given time To generate different views of the same image set use the Duplicate function in
452. scence images from raw images to obtain Rkorr F1 F1 Background F2 F2Background when calibration reference is not obtained with the experimental sample in situ e Calculate ratio R e Perform equation or titration calibration compare R with a calibration curve gt titration calibration or put R values in calibration formula Raw Images SPECE e Background and Autofluorescence image pair a ee i Erini eee ei L Ra cr gt Egan mr Pate nee i Th F ff iir z p in Sa FP tinea Scaling of calibrated image J Ppacwitreye r Fig 4 138 Ratiometric Dyes Calibration 4 144 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Process Menu Carl Zeiss 4 6 12 7 Ratiometric Dyes Equation Calibration H C Single Wavelength Dye Calibration Grynkiewicz f Ratiometric Dye Fmas R Amin Conc Kd 3 i E Fura 2 Indo 1 2 cee ie Dama Kp dissociation constant taken from literature z een nming Fma s2 20 00 Rmin derived from ion free state of the dye e g O In Vitro Amin 0 20 Click into window ace Ca2t en Fmin 200 00 Rmax 4 00 Click into wind Rmax derived from ion bound state of the dye _Chk irto window e g saturated with Ca2 Fmin2 and Fmax2 are the minimum and maximum Y Fig 4 139 lon Concentration button fluorescence intensities at wavelength 2 Rmin Rmax Fmin2 and Fmax2 may be determined in th
453. sdoosousol _ BuiuueosoGs ywuseser UYLISONGd JALIT S NSI JA 7 S WSI March 2006 INTRODUCTION LSM 5 LIVE Carl Zeiss LSM 5 LIVE DuoScan Knowledge of this manual is required for the operation of the instrument Would you therefore please make yourself familiar with the contents of this manual and pay special attention to hints concerning the sate operation of the instrument The specifications are subject to change the manual is not covered by an update service Unless expressly authorized forwarding and duplication of this document and the utilization and communication of its contents are not permitted Violations will entail an obligation to pay compensation All rights reserved in the event of granting of patents or registration of a utility model Issued by Carl Zeiss Microlmaging GmbH 07740 Jena Germany Phone 49 0 3641 64 3400 Fax 49 0 3641 64 3144 E mail micro zeiss de www zeiss de Ism B 45 0019 e 03 06 LSM 5 LIVE INTRODUCTION LSM 5 LIVE DuoScan Carl Zeiss How to make best use of the LSM 5 LIVE operating instructions A few symbols in these operating instructions will help you to recognize the nature and purpose of information immediately A A A A The WARNING symbol warns against hazards for the user that might arise when operating the laser This WARNING symbol warns against hazards from dangerously high voltages The CAUTION symbol warns against faults and hazards that might arise
454. select a microscope configuration e Click on the Apply button A new button with the name of the selected microscope contiguration has been created e Click on the Close button to close the Assign Microscope Settings To Button window e Click on the Close button to close the microscope window For the conventional setting of the Axiovert 200 M proceed as follows e Click on the VIS button in the Acquire subordinate toolbar e Place specimen on microscope stage The cover slip must be facing down e Inthe Objective list box select the required objective e Use the focusing drive 4 32 4 to focus the required specimen plane e Select specimen detail by moving the stage in X and Y via the XY stage fine motion control 4 32 3 and 2 4 48 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE Fig 4 32 OPERATION DuoScan Acquire Menu KR WN Focusing drive Lh ek LSM 5 LIVE with Axiovert 200 M BP Microscope settings on Axiovert for transmitted light observation e Click on e Click on set the the Reflected Light button and set the shutter to the Closed position the Transmitted light button Click on the On button in the Transmitted Light panel and transmitted light intensity via the slider or click on 3200 K Click on Close to close the Transmitted Light panel e Click on the Condensor button and set the aperture via the slider in the Condensor panel Set the filter in the Filter selection box
455. ser Linesj Help75 window and to check the settings you made The window is closed via Close 4 52 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss All amendments made in the Configuration Control or Laser Control window are updated on line in the Detection Spectra amp Laser Lines window e Aclick on the Laser button enables you to open the Laser Control window switch lasers on and off if required and control the laser output 4 5 3 3 Config Button oe Mame FITC Rhod The Config button permits existing track configurations to be loaded stored under any Fig 4 36 Track panel name or deleted 1 Load a track configuration TH Track Configurations store Apply Configuration A contiguration stored in the system whether factory supplied or user created can be accepted or selected for active operation as follows e Click on the Config button the Track Configurations window appears on the Fig 4 37 Track Configurations window screen Configurations e On the Store Apply Configuration panel click on the arrow button Ba This opens a list box of all stored track configurations e Browse through the configurations by clicking or use the scroll bar at the side of the list box e Click on the desired configuration The selected configuration is shown in the first line of the Configurations list box e g FITC Rhod e Click on the Apply button This resu
456. sity ratios of two input channels may also be used for F FO calculation e g with single wavelength ton indicators Fig 4 58 Channel Settings panel of a Ratio Channel Laser Power Z sem Ss fe al samm 30 A gt gt F xm or gt Line active Transmission Fig 4 59 Excitation of Track panel 03 06 B 45 0019 e 4 77 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Acquisition of a frame Once you have set up your parameter as defined in the above section you can acquire a frame image of your specimen Channel Settings e Click on the Single button in the Scan Control window The system will automatically start the acquisition of a frame The individual channels and the overlay image can be viewed by changing to the Split xy mode This button is located on the right hand side of the Image Display window Channels Pinhole 2 Optical slice lt 14 9 pm Pinhole 1 03 Airy Equival The following scan image shows the result with Detector Gain 1 A two defined tracks and the overlay see Fig 4 61 See the appropriate Channel Settings panel in heen 0 gt the Scan Control window Fig 4 60 one ime 2 4 S e 0 9 Frames per Second Fig 4 60 Channel Settings panel for two defined tracks plus Ratio channel Ist track 2nd track CALI T1 CALZ T 2 ee OHLI T 2 CHL2 T
457. spline track in space Fig 4 278 Render Series window e g Position List mode 4 276 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 Time series When the input images is a Z Stack time series the reconstructed images are generated for each time point e f you click on the right mouse button in the main display window of Image VisArt two options appear Render properties Renderer This functionality helps to find the optimal performance when working with different hardware renderers and to speed up some advanced rendering modes OPERATION Display and Analysis of Images Carl Zeiss 3D Renderer Renderer C OpenGl Software GOI Generic Microsoft Corporation Version 1 1 0 Cancel GDI Genernc Microsoft Corporation Version 1 1 0 C Direct Software Microsoft Reference Rasterizer Software Vertex Processing Version 8 i DirectSD Hardware Rasterizer ATI FireGL 3250 Software Vertex Processing Version 5 C Direct3D Hardware ATI FireGL 3250 Hardware Vertex Processing Version 3 Render board features to use Palette textures for single channel images Intensitysdipha textures for single channel images Iw textures only Enable display lists Use up to 197 8 MBytes for Textures Default Fig 4 279 3D Renderer window 03 06 B 45 0019 e 4 277 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 24 Displ
458. ss Objective Name hew objective n Objective ID PO Magnification o1 O Apply Aperture 1 Imersion Medium Refrective Index 1 Working Distance oo UV Fitness no xl Scan Fitness Fig 4 222 Create new Objective window e To remove an objective from the database select it with a click of the mouse in the Change Objective panel and then click on Remove Objective The objective is removed trom the list e Click on Close to close the Remove Objective window 3 Edit Objective You can only edit objectives in the User Defined Objectives directories e To edit an objective from the database select it with a click of the mouse in the Change Objective panel and then click on Edit Objective The Edit user defined Objective panel will open and the parameters of the Objective can be edited e Click on Close to close the Edit user defined Objective window 03 06 Edit user defined Objective Producer Objective Name Objective ID Magnification Aperture Imersion Medium Refrective Index Working Distance UV Fitness Scan Fitness Fig 4 223 B 45 0019 e Carl Zeiss Jena GmbH Won Zeiss Close Plan Neofluar 9999 00 55 Apply Edit user defined Objective window 4 213 OPERATION LSM 5 LIVE Carl Zeiss Maintain Menu LSM 5 LIVE DuoScan Objective Control 4 10 2 2 Focus Speed Change e Change the required objective in the nosepiece Tn e Click on the Objective button
459. st of Tracks Switch tracks after each es Frame Fast Name Channels Light nm I Track ChL1 455 E Track ChL2 405 a Add Track Remove Store Spply Single Track k Beam Path and Channel Assi nment The maximum of four tracks with up to 8 channels can be defined simultaneously and then scanned one after the other Each track is a separate unit and can be configured independently of the other tracks with regard to channels Acousto Optical Tunable Filters AOTF emission filters and dichroic beam splitters e Select Channel Mode if necessary Fig 14 LSM DuoScan LSM 5 LIVE l LP 505 EE E m ChI Ez EF 415 480 NFT 490 F4 l _ r Che A Excitation T 488 30 F4 Specimen Fig 14 Configuration Control window for Multi Track 03 06 9 e Click on the Multi Track button in the Configuration Control window Fig 14 The following functions are available in the List of Tracks panel Fig 14 Add Track button Remove button Store Apply Single Track button 1 J An additional track is added to the configuration list The maximum of four tracks can be added One track each with basic configuration is added i e one ChL 1 channel is activated all laser lines are switched off emission filters and dichroic beam splitters are set in accordance with the configuration last used The track previously marked in the List of Tracks panel in the
460. sting image to be copied and stored as a new image The selection of Source Channel and Destination is made in the same way as in the Add function see Add page 4 121 4 6 5 1 03 06 Open Close the Copy Window Click on the Copy button in the Process Subordinate toolbar of the Main menu This opens the Copy window Click on the Close button to quit the Copy window B 45 0019 e OPERATION Process Menu Carl Zeiss Operators and pre defined vanables x Operator Aliases Precedence _ aon mad ELSE CSUN Close z8 2 src arc sree source source source negi negate 1 not not a P A powl power ee E Insert roull noultiplyt 3 an i div dividel 3 Es E modi ref remainder 4 addi 4 o subf subtract 4 reater atl 5 lt z amallerj lezs ItL z b ek and andl E B xor wor 7 L oror E _condi conitionall select J T mini _ minimum maf _masimumf abs Fabal fabs qr ae root zini ainel cosl cosine E tan Tooo asni arcsin asinel arcsine atant arctanll atannanki l archos Destination image d represents the intensity value that i written to the destination image for the pixel If the Absolute intensity mode is switched off the value is scaled from range 0 1 to the range O maxinunn intensity for the data type of the channel in th
461. t 145 497 kBute Clipboard 337 063 kByte Fig 4 12 Database window Gallery display mode In the Options menu in the function Settings it is possible to define the start mode of the image database Form Gallery Table the recordset displayed first last middle and the parameters shown e To select one of the images of the database for normal size presentation double click on the desired image The same can be achieved by clicking on the desired image in the gallery and then clicking on the Load button e To select several single images press amp hold down the Ctrl key and select each desired image by a click of the mouse If several images have been selected they will all be opened and displayed one after the other e To select a number of consecutive images press amp hold down the Shift key click on the first and the last image to be selected All the images between these two will also be included in the selection It several images have been selected they will all be opened and displayed one after the other Selected images are highlighted in blue Furthermore the current image selected last receives a patterned frame 03 06 B 45 0019 e 4 23 OPERATION LSM 5 LIVE Carl Zeiss File Menu LSM 5 LIVE DuoScan 4 4 3 3 Table Display Mode e Click on the Table button All images of the image database e g Convallaria mdb image series are shown in Table display mode on the screen amp Convallaria
462. t toolbar In addition the Animate window appears in i 7 I which you can intluence the direction and speed of Fredy 5120 512 8 e 3D image rotation see Animate page 4 239 Fig 4 153 Projection image You can browse through the rotation sequence manually with the Slice button in the Select toolbar and the Slice slider e To view the computed 3D sequence as a gallery on the screen click on the Gallery button in the Display toolbar Projection Convallariaz AlM Display Ready 12 512 176 3 channels 8 bit Fig 4 154 Projection image Gallery 4 158 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan 3D View Menu Carl Zeiss 4 7 3 Stereo Stereoscopic images can be generated in a variety of ways by means of the Stereo function A stack of images must be available 4 7 3 1 Open Close the Stereo Images Window e Click on the Stereo button in the 3D View subordinate toolbar of the Main menu This opens the Stereo Images window e Click on the Close button to quit the window 4 7 3 2 Source Panel e Select the image for the projection operation from the image selection box e Select the channel to be used from the Channel selection box Stereo Images Is Click into window aw Pe Stack chs H Stereo Images Projection Transparency Mode Red Green Image Split Images Basic Angle fo E pz RightLeftAngle 12 E EA E Number Images 2o
463. t Size oh lysed i Input 2 OK Cancel POA Fig 6 9 This operation is useful to add a segmented channel or any other result of a function to the original image sequence The selected channels of Input 1 and Input 2 are copied to Output The maximum number of channels in an image sequence is eight If the image sequences do not have the same extents Output Size defines which input is taken as a reference This selection also defines the properties for scaling and units in the output image sequences Parameters Input 1 First input image sequence Input 2 Second input image sequence Output Output image sequence Output size Defines source image sequence for size scaling and units 03 06 B 45 0019 e 6 15 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan On the Delete Channel tab sheet channels of the Input 1 image sequence can be selected to delete channels Edit Channels Input 1 hooo MER Output 2 New Cancel Apply Fig 6 10 This operation might save time and memory for further processing if not all channels are needed Only the selected channels of Input 1 are copied to Output Parameters Input 1 Input image sequence Output Output image sequence Delete All Images This function deletes all images and image sequences from the memory Gallery The function is used whenever a completely new image sequence should be processed In order to drop the images item by item to the w
464. t Tit fit procedures plane cylinder sphere inverse and fill holes 4 308 B 45 0019 e LSM 5 LIVE LSM 5 LIVE DuoScan 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 25 Display Prev This function allows to compose images graphs and text for printing use any image format change fonts and line width in graphs via context sensitive menus The settings of Chan Zoom Slice Contr and Palette apply In the Options menu in the function Settings with the tab Print Status Display parameters are determined and the Print Status Information is activated deactivated Click on Prev will display the Preview window and the Print toolbar Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily deleted while the toolbar is displayed Display Print Print Setup 1 Info El Ready Ale wale 2 channels 3 bit Fig 4 301 Image Display window Prev display with Assembly of image intensity profile and scan info 03 06 B 45 0019 e 4 309 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 25 1 Context Menu for Scan Information Text The context menu right mouse button allows to vary the output of the scan info e Click with the right mouse button A context menu with the options Color and Font is displayed e In the Color menu you can select a different type color f
465. t possible off line mode e Start Use of this button starts the software system OFF LINE Initialization After the start instrument initialization is performed and can be monitored in the Initialization window and interrupted with a click on the Cancel button if required Initialize CF Stage Depending on the selected option Online Mode Images or Offline Mode initialization is performed in the offline or online mode Fig 4 4 OFF LINE Initialization window LB Some printers for example KODAK Thermo Printer will produce an error message hard key not found in case the printer is not switched on Remedy turn on the printer before starting the LSM 5 software Don t switch off the KODAK printer during the scanning process 4 14 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Main Menu Carl Zeiss 4 3 Main Menu The major functions can be selected in the Main menu either via the pull down menus in the menu bar or via the Main menu toolbar which can be displayed or removed as required Further subordinate toolbars are available below this toolbar depending on which button has just been pressed File Acquire etc In the standard setting of the LSM 5 software the toolbars are automatically displayed after clicking Start L However since the LSM 5 software is operated more conveniently with the help of the toolbars only this method of function activation will be described in the following e
466. tach the LSM DuoScan to or from the microscope stand Fig 1 14 Fastening screws and connections on LSM DuoScan To ensure functioning of the system and laser safety the following connections have to be changed 1 The connection of the microscope to the safety interface of the system is located either on the additional Safety Box Axioskop 2 FS MOT Fig 1 15 1 or on the rear side of the microscope Axiovert 200 M or Axio Imager Z1 Fig 1 16 1 and Fig 1 17 1 This connection has to be unplugged from the microscope which is not in use and plugged into the microscope to be used following the exchange of the scan head Fig 1 15 Safety Box of Axioskop 2 FS MOT Fig 1 16 Connection of Axio Imager Z1 with main connection to safety to safety interface 1 and interface 1 electronics 2 03 06 B 45 0019 e 1 23 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss User Interface LSM 5 LIVE DuoScan Fig 1 17 Connection of Axiovert 200 M to Fig 1 18 Four CAN connections 1 are safety interface 1 available on the rear side of the electronics module The microscope in use has to be connected to one of them 2 The plug for the main connection of the microscope to the electronics is situated on the rear side of the electronics rack It is either of the four CAN connections shown in Fig 1 18 Only ONE microscope can be connected to the electronics at a time The connection has to be plugged in and unplugged at the electronics rack Th
467. te Frames per Second 03 06 B 45 0019 e 4 75 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan Scan Control Gain and Find The Find button always sets an ideal gain for a clear background best sensitivity and full grey resolution This is maximally a gain value of 25 One can manually increase this gain by moving the gain slider beyond 25 and use the digital post amplification by doing this Be aware that from value of 37 5 on background artefacts can be visible since those are amplified too r E E i When using 12 bit grey resolution gaps can occur cute Ep PE Mex f Sige in the histogram of the acquired image in case the Gain slider is moved beyond a gain level of 25 Detector Gain Amplifier Offset Use the Amplifier Offset slider to remove underexposed pixels in the image background no blue pixels visible with range palette Poel Time 30 32 psec Scan Time 16 66 meee Fig 4 57 Gain and Find L gt The parameters Detector Gain Ampl Offset and Ampl Gain are described in section Confocal Aperture Detector Gain Ampl Offset see page 4 319 in the context of image optimization 4 76 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan The parameters of a ratio channel are set in a separate dialog box e Click on the button of a ratio channel e g R1 The dialog box for the setting of the ratio parameters is displayed Clicking on the required tabs enables you to choose f
468. te List panel File with the extension lut are LSM 310 410 palette Tiles 4 13 9 Select Anim Animate Close This function allows to animate frames of a Z Stack or a time series specify animation parameters such as range and animation speed Click on Anim will display the Animate toolbar Any changes done with this toolbar are effective Bea immediately Speed z 10 When the image s displayed in the Image Display T a window is neither a Z Stack nor a time series this l button Is grayed and not accessible Fig 4 251 Animate window e Click on the Anim button in the Select toolbar of the Image Display window of a stack The Animate window will be displayed and the animation started immediately e Click on the Close button to close the Animate window and to stop the animation 03 06 B 45 0019 e 4 239 Carl Zeiss OPERATION LSM 5 LIVE Display and Analysis of Images LSM 5 LIVE DuoScan The animation is controlled via the following function elements Current Slice Start End a P Speed 1 Speed 2 up Increment 4 240 Current Slice slider Manual movement through the individual slices of a stack by moving the slider or by entering the slice number in the input box Slider can be accessed only when the automatic animation is off Start slider The setting of the Start sliders limits the number of slices to be used for the animation Previous slices are not ta
469. te the image to be shown in full size by clicking on the image content e Click on the term Window in the menu bar of the Main menu The Window menu pull down will be opened e Click on the Full Screen line The image will be displayed in full screen size e Click in the image to show it again as an Image Display window in normal size 4 11 2 Close All Image Display Windows This function closes all the opened Image Display windows e Open the Window menu e Click on the Close All Image Windows line All the opened Image Display windows will be closed Carl Zeiss In the Options menu in the function Settings in tab Save at position Save prompt at closing modified windows it can be determined whether a prompt is shown on Closing of All Image Display Windows or not 03 06 B 45 0019 e 4 221 OPERATION Window Menu Carl Zeiss Pile E3 Objective Plan N ecfluar 10 0 Pixel Depth E bit Stack Size pm 637 05 um s 637 05 pri Stack Size Pixel 51 eu Ol Scaling 1 24 um 1 24 pm A Scan and System Information Stage Position x 0 00 pm of 0 00 pm lt 0 000 pm Average fi Next Scan Plane OSMByte Time 2 5 Mbyte Rak 5s ME fee D Pagefile 21 61 ME free B E patem mode LSM Time ji rear 2006 09 25 48 4M lee Image Memon RaM Disk amp j Fig 4 234 Scan Information window 4 222 LSM 5 LIVE LSM 5 LIVE DuoScan 4 11 3 Scan and System Information This window displayed
470. tector Gain until the red pixels only just disappear 03 06 Pami ODE a M arene WD r sc a i Ds T Pia Plier Leathe te Fig 18 Image window Color Palette Color Palette List Mo Palette Glow Scale Rainbow Rainbows Add to List o Da Fig 19 Color Palette window Pinhole a dro m gt 1 Mas mi Optical slice lt 14 9 pm Pinhole 1 03 Ain Equival Detector Gain E _ gt Amplifier Offset o 4 e gt enn Time 2 ip 4 i 0 5 Frames per Second Excitation Line active Transmission ana A M 4sanm 5 d F nho M 532mm 30 F C 633nm 01 4 gt gt Fig 20 Scan Control window Channel settings TS Scan Control Fl x Use T eee i tre rane P Dod q E Stack Size 19 64 pm en Focus 0 00 pm en Sectioning Mark First Last Num Slices E 4 a m gt Fast vy Interval um 393 E gt KA Current Slice 6 4 p cl Start Fast Stack Keep Interval Keep Slice STOP Stop Move to r il Las Ret Cor 1 Ado H i AyZ ev cont Auto Z Corr Ex Auto ied foe 2 Settings Fig 21 Scan Control window Z Stack settings Scanning a Z stack e Select Z Stack in the Scan Control window e Select Frame if necessary The Z Settings panel appears e Select Mark First Last on the Z Settings panel e Click on the XY cont button A continuous XY scan of the set focus position
471. tem PC switch to OFF position Fig 1 e Put the MAIN SWITCH on the system rack to OFF position Fig 1 e Switch off the HBO 100 mercury lamp Fig 2 03 06 15
472. ter cera FA Pane kepsen a a a A 4 129 PrE eW P lae S a N decent ee det oaaces aaeereaece tee 4 132 COME OSU rnrn a a a a eco ieoattoon 4 132 IREE ole fe 10 PE PEN EN EIE EE TAEA ES PEPE O AE TA E ee ee TE 4 133 Open Close the Interpolate Brightness and Contrast WiNndOW sss sessisesiieesireerirerrnn 4 133 ACO Ea EEE E A A ATA E E E AE 4 133 Menpan PANG lea a a a tend ob eetlci shales a 4 134 PREVI CMV PING lenses cenit imate r shot 5 ohte et Luda 4 135 ENI EE EEEE aaa ante vee E maaan cette E A E A ETS 4 135 Open Close the Channel Shift WiNdOW eessnsssssriusssriussrrressrrrrerrirerrrrrerrrrreerrrn 4 135 maade P anae eaea E E T E O 4 136 SA a Met ae E E TE cs A E AE E E AAE AEE 4 136 POVE ING lene E oe hale haut E T S 4 137 I a a a a aa e a teat cme antasee sn oeetabacnact 4 137 Open Close the Unmix VIN GOW oiskc Bi sariciah ite sbitonantck tac Gcbiattniniadicneaansbonkinieniaild 4 138 SUE PUR EEES AE AE E eran etna EES ANVE E 4 138 Definiti n of Channels Pane lheit nadani dain 4 138 FOU ONGC UO saa ea a a a anelioshateehemoaie 4 141 Open Close the lon Concentration WINdOW sss sssiisssirssriuesrrrerrrrerrrrerrreerrrerrrre 4 141 FOC ION 1 SS Gil HOI ass a E A AE 4 141 B 45 0019 e 4 3 Carl Zeiss 4 6 12 5 4 6 12 4 4 6 12 5 4 6 12 6 4 6 12 7 4 6 12 8 4 6 13 4 6 14 4 6 15 4 6 16 4 6 17 4 6 17 1 4 6 17 2 4 6 17 3 4 7 4 7 1 4 7 1 1 4 7 1 2 4 7 1 5 4 7 1 4 4 7 2 4 7 2 1 4 7 2 2 4 7 2
473. th this function are effective immediately The overlay graphics can be stored together with images and can be retrieved from the LSM 5 image database e Click on the Overlay button in the Select toolbar The Overlay toolbar will be displayed on the right hand side of the Image Display window e f you click on the Overlay button again the Overlay toolbar will be removed Provided that the display of the overlay elements has not been deactivated by clicking on the Off button the created elements will still be displayed in the Image Display window even after closing of the Overlay toolbar Convallaria AIM a BO pr A 9056 Urn amp 7841 75 um x Ln 35 76 um 69 6 k 39 feum 475 57 45 Um Ready 51225128 20 3 channels 6 bit Fig 4 242 Image Display window Select Overlay 4 230 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 6 2 Buttons in the Overlay Toolbar The following functions can be used on activation of the buttons in the Overlay toolbar S Arrow selection button Activation of the mouse button for selection resizing or movement of an overlay element in the Image Display window Resizing Click on the handle and hold down the mouse button drag the handle release the mouse button Movement Click on the line and hold down the mouse button move the entire element release the mouse button Line butto
474. th unique technological performance It is made of high precision components representing latest technological developments and a design that was guided by ergonomics best possible performance and every day usability The system is highly modular and flexible but also easy to service and open for upgrades 2 2 System Components and Recommended Setup The core of the system is a vibration isolating air damped microscope table with a breadboard for fixation of the system and possible accessories We strongly recommend the use of our Science Desk tables since these have the most compact size that allows the setup of the various system configurations and future upgrades A wide range of special accessories for physiological molecular or cellbiological or technical experiments e g posts shelves grounding faraday cages cabinets etc is available worldwide by our partner Melles Griot The science desk is a professional open system platform for your experiments A wide and a narrow version of the science desk table is available matching the side or rear port configurations of the microscope stands The microscope table is complemented by the laser and electronic system rack which can be positioned at the side or at the back of the microscope table This rack contains all electronic and laser components the LSM 5 LIVE needs and is again designed with best performance usability and future upgrades in mind For setup and service purposes free acc
475. the additional functions are no longer displayed and on closing the Brightness and Control window 4 13 7 2 PolyLine The intensity is set in the Intensity Screen via a freely selectable number of knots which permits the creation of an intensity line in the form of a polyline The number of knots can be selected from the Number of Knots selection box rs The original line form is reset via Reset The line form will be retained even when the additional functions are no longer displayed or when the Brightness and Control window is closed OPERATION Display and Analysis of Images Carl Zeiss Brightness and Contrast Result Intensity Screen Intensity in Memor ee ee i a n a Ramp PolpLine Spline Gamma Reset Fig 4 245 Brightness and Contrast window with activated Ramp function Shape Result Intensity Screen Intensity in Memory te W m ell Ramp FolyLine Spline Gamma Reset Number of Knots E Fig 4 246 Brightness and Contrast window with activated PolyLine function 03 06 B 45 0019 e 4 235 OPERATION Display and Analysis of Images Carl Zeiss Shape Result _ Intensity Screen Intensity in Memory Ramp Polpline j Fig 4 247 Brightness and Contrast window with activated Spline function Shape Result Intensity Screen Intensity in Memory Fig 4 248 Brightness and Contrast window with activated Gamma function LSM 5 LIVE
476. the Process menu Rs During image acquisition all active display functions can be used 4 13 3 Select Chan This function permits to change the color assignment of channels of images switch individual channels of a multi channel image on off switch to monochrome display of the image instead of color display e Click on the Chan button in the Select toolbar The Channels toolbar will be displayed on the right hand side of the Image Display window IB Any changes done with this toolbar are effective immediately e Click on the Chan button again to remove the Channels toolbar Convallaria AlM Display Channels Ch F eadp 51248512420 3 channels 5 bit Fig 4 237 Image Display window Select Chan 4 226 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 3 1 Assigning Another Color to a Channel Lor e Click on one of the channels button in the Fig 4 238 Color selection box Channels toolbar e g Ch1 The color selection box with all the currently defined colors will appear e Click on the required color The selected color will be assigned to the current channel the color selection box is closed and the displayed image is updated The control box of the channel button e g Ch1 also shows the selected color 4 13 3 2 Switching a Channel of a Multi Channel Image off or on e Click on one of the chann
477. the Time button the recording of the series will not be definitely finished It is possible to either continue the series via new settings of Trigger and Time or to definitely finish the time series via the Stop key The following example of a scanning image was taken using the Time Series function Both the time and the markers set during the scanning procedure are projected in the image series in different colors If the cursor is moved to a marker position in the scanning image the relevant information on the image detail is automatically provided in an additional window 4 102 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Colored marker position Repke Sis Sizal e J0 i cheret Ehi Fig 4 88 Image Display window of a Time Series Scan The image markers have different colors with the following meaning e red manually set marker with time indication and comments e blue automatically set marker with change of delay 03 06 B 45 0019 e 4 103 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 6 7 Time Series of a Frame over Z Stack option e First set all parameters required for recording a Z Stack in the Scan Control window e Then set the parameters required for recording the time series in the Time Series Control window identical procedure as for the time series of a frame e Start the time series by clicking on Start T Complete stacks are
478. the channel button e g Ch1 or influence all channels simultaneously by clicking on All e Clicking on the Reset button will reset the Original setting of brightness and contrast Brightness and Contrast e Clicking on the Close button will close the Brightness and Contrast window Further contrast and brightness parameters can be activated or deactivated alternately using the More and Less buttons Fig 4 244 Brightness and Contrast window e Click on the More button to display the additional functions The Brightness and Contrast window will be enlarged the labeling of the button changes from More to Less If you click on Less the additional functions are no longer displayed Simultaneously with the setting of brightness and contrast the intensity values of the image can be set directly in the Intensity Screen via the Ramp PolyLine Spline and Gamma functions The intensity values can also be set either for all channels together or individually If the image has already been changed using the Contrast and Brightness sliders this setting difference is displayed in the Intensity Screen by means of the Shape and Result lines 4 234 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 13 7 1 Ramp The intensity is set via two knots in the Intensity Screen which allows an intensity line to be created in the form of a ramp 3 The original line form is reset via Reset The line form will be retained even when
479. the parameters in the same way as for Time Series of a frame e Then click on the Mean ROI button in the time series frame A mean intensity profile of the defined ROIs To be defined using the Edit ROI see page 4 90 is created as a function of time Unnameds AIM iof x Intensity ROI 1 Select Display Scan Mean of ROIs 250 E Of 200 Chan xy Hi Type Position Dimension 150 PE All ARM gat a coment Zoom Spite 1 CO 149 114 127 121 100 ta EEE IV 2 Cy 379 126 184 175 m cipe Fallen Vv 3 O 369 326 120 130 0 Overlayi Hist f 0 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 40 P Scaling Automatic Time Range Number Cycles Sj r s a o oc 8 85 8 D al e T Jg o i shale T T au Nm lt a T a Th lt J l to E a a aS ra a Lle i 0 0 0 5 1 0 15 2 2 5 3 0 3 5 4 0 Time seconds Copy Image Table Show Image Sopp Table Show Table i es ee es ee 2 o 6 8 8 8 6 on ow ag 0 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 Time seconds R eady z S5can 1x385 1 channel Fig 4 90 Image Display window of a Time Series with Mean ROI The Image Display window of the Mean ROI function is structured differently than that of a frame On the left hand side of the Image Display window the intensity time profiles per ROI are displayed graphically The Select and Display toolbars which are also available in the standard Image Display window are positioned in th
480. the unit for Mean of ROls diagrams 4 9 2 12 Mean of ROIs The Mean of ROls tab permits the presetting of the Image Display window for the optional MeanROI function time series to be changed with regard to scaling and display mode of the intensity time diagrams 1 Diagram Scaling The following settings are possible by activating one of the option buttons Automatic diagram scaling OPERATION Options Menu Carl Zeiss petnenenanenenananensesaseneneeeeeeeeesnsensnsssssssssssssnseses Recording Reuse Timeseries T Temporary Files Scan Mean of ROIs Use the following Parameters for Image Acquisition after Apply Recording Configuration NM Objective IV Average MV Frame Size IV Pinhole Diameter MV Speed V Detector Gain and Offset J Data Depth I Zoom J Scan Direction IV Rotation and Offset Set Objective for Reuse Fig 4 210 Recording Reuse tab Recording Reuse Timeseies ScanMeanofROIs Tempora Fies m Time Control Cycle Delay C Time Interval Unit for Mean of ROIs diagrams Seconds Minutes C Hours Fig 4 211 Timeseries tab Recording Reuse Timeseries Scan Mean of ROIs Temporary Files Diagram scaling C Automatic diagram scaling Fixed time range for diagram time scale Fixed number of cycles for diagram time scale Range 20000 m Initial diagram types Live image Dne diagram MV Black graphs Channels d
481. tical axis of the microscope corresponds to the zero position of the piezo stage i e to the center of the specimen holder in the stage tongue Then perform initialization by pressing the piezo Null button This step must be repeated after every new Start of the system Also see the notes on the operation of the motorized scanning stages If the system is equipped with a manual microscope stage the user has the option of performing the calibration by entering the ex shift in mm via the Calibration slider The shift is read off from the microscope stages In the case of the manual Axiolmager stage x can be read directly from the scale adhered to the front of the stage In the case of the manual Axiovert 200 M Stage a scale is located on the right of the knob where the 45 mm ex shift relative to the zero position of the microscope stage can be read off The ex value Is positive for both stages if shift from the zero position is made to the right and negative if the shift is made to the left On account of the inclined position of the stage tongue the object is also shifted laterally during the fine focusing motion This lateral shift is negligibly small if as recommended by us specimen carriers with thickness 1 0 mm are used exclusively Otherwise the marked lateral shift of the object during fine focusing can result in image distortion For the same reason Petri dishes without fixation ring must be used exclusively The nosepiece of the Axio
482. tion panel The stage moves to the selected position e Then click on the Single button in the Scan Control window to scan the selected area as a single image The single image is scanned and displayed in a new Image Display window 3 Overlay functions cannot be activated in the Tile Scan Image Display window The created overview frame can then be stored like any other scan image If a stored overview frame is opened again the rectangle with target will appear again However it can be deleted using the Overlay function 03 06 B 45 0019 e 4 119 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 10 VIS TV and LSM Buttons The VIS TV and LSM buttons are included in the Acquire subordinate toolbar of the Main menu They switch the beam path and indicate which beam path has been set in the binocular tube of the microscope VIS observation via the eyepieces of the binocular tube lasers are off TV camera observation if connected via camera adapter of the binocular tube LSM screen observation via laser excitation using the LSM 5 LIVE and software evaluation rs Ifthe beam path of the microscope is changed manually via buttons on the tube this is recorded by the software and the relevant button is activated automatically Vice versa the beam path can be switched via activation of the appropriate button in the software 4 120 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Pro
483. tion Blocks y This block has to be used if the actually scanned image s should be displayed F Image Display For each Image Display block a new image window is opened If not saved the image will be overwritten when the program flow is passing by again V Q lt x rr O Q Q gt A et O O A at N The image s are stored to a image database The database taken is either the l Se eae last one that was open or the current open one Clicking Read back updates the database to be used for storing the image s In addition some other parameters can be set according to the list of properties displayed Within the program flow already existing images can be loaded Y v The database taken is either the last one that was open or the current open one Clicking Read back updates the database to be used to load the image s from The images can be loaded by Index or by the name of the image It is possible to export images choosing a certain file format from the pull down list that appears when highlighting the line File Format in the properties list Specific parameters are assigned to each file format which can be set for the image export R 4 Images can be imported within the program flow The image to be loaded can be selected by typing in the file path The import of single images or a image series is possible The images to be imported as series must have the same name and must be numbered in ascending order
484. tion button you can check at any time which lasers are available for active operation If you deactivate Line Active the laser wavelengths for the lasers are deselected by means of the attenuator i e these lasers change into standby status Excitation filters emission filters Achrogate main beam splitters and NFT secondary dichroic beam splitters can be switched online channels PMT photomultipliers only off line 03 06 B 45 0019 e 4 59 Carl Zeiss T Configuration Control Channel Mode sie ack Track hluti Track Ratio E List of Tracks Spectra Frame Frame Fast Tight i Track Track Switch tracks after each Add Track Remove Beam Path and Channel Assignment LSM OuoScan LSM 5 LIVE l LF 505 a rm ChI BP 415 480 MFT 490 A A A pcan T 468 30 Z Specimen a Fig 4 43 Configuration Control window Multi Track activated 1 Beam Path and Channel Assignment panel OPERATION Acquire Menu Store Apply Single Track Contig LSM 5 LIVE LSM 5 LIVE DuoScan 4 5 3 5 Settings for Multi Track in the Channel Mode The Multi Track function permit several tracks to be defined as one configuration Recording Configuration for the scan procedure to be stored under any name reloaded or deleted The maximum of four tracks with up to 8 channels can be defined simultaneously and then scanned one after the other Each track is a separate unit and can be contigured independently of the other
485. toolbar opens the Cut to the right of the Image Display window 1E Any changes done with this toolbar are effective ereen immediately The content of the overlay plane is 258 256 g 19 E 3 ii temporarily deleted while the toolbar is displayed Mid Mid Mid of oO Reset All Trilin ar Interpolation e By varying the parameters X Y Z Pitch and Yaw you can position a section plane of any orientation within the stack volume View of section planes e The resulting position of the section plane is shown as a red area below the Trilinear Interpolation button At the same time the result is shown in the Image Display window e A click on the Reset All button restores the original position Fig 4 258 Image Display window Cut display e A click on the Trilinear Interpolation button will improve the quality of the image by performing a 3D interpolation of the image 4 250 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 19 Display Gallery This function allows to display images Z Stack time series combination of both side by side in tiled fashion add data relevant to the images displayed Z Stack slice distance time of acquisition or wavelength extract a subset of images trom the original stack and store the result as a new image The settings of Chan Zoom Slice Contr and Palette apply Click on Gallery will display the Gallery to
486. transferred to all the other windows concerned 03 06 B 45 0019 e 4 9 OPERATION LSM 5 LIVE Carl Zeiss Purpose LSM 5 LIVE DuoScan 4 1 2 Windows and Window Elements Window element Description Explanation 14 Laser Control x Window e g Laser Control window a Window displayed after activation of a z LIOS6 i Laser Unit Wavelength function button e g Laser button in the b a toolbar of the Main menu Compass 315M Aa nm Sapphire 486 sapphire 456 Maximum Power 100 0 mw Wavelength 496 nm Status Ready Tube Current 00A Standby Lasers Panel e g Lasers panel wanie Limited function range within a window Toptica 405 50m 405 nm Toptica 639 639 nm Compass 315M Faz nm Sapphire 488 466 nm Laser Li HEE IL List box or selection box Toptica 405 50m 405 nm Toptica 639 639 i i i Compass ISM ED nm i Selection of one of the displayed options at a Sapphire 488 436 rim click of the mouse Open the box by clicking on the arrow Filter EP 505 550 button 25 Input box Input of text or numeric values via the keyboard 25 H _ 5 Scrollbar with slider Setting of numbers in the relevant input box by moving the slider or clicking on the arrow buttons or clicking on the slider and moving via the arrow keys of the keyboard Press the Shift or Ctrl key while clicking on the arrow button to change the numeric values in coarse or fine steps
487. tter ON OFF with indicator Light intensity control Focusing drive Control knob for X travel of mechanical stage Control knob for Y travel of mechanical stage Buttons for switching the beam path TIN AA QS ome Fig 4 29 LSM 5 LIVE with Axio Imager Z1 Microscope settings on Axio Imager for transmitted light observation Set the reflector turret position to None and click on the On button for transmitted light Control the brightness of the halogen lamp with the potentiometer 4 29 4 or the Intensity slider in the Transmitted Light panel Set the required transmission value of the gray filters in the Filter frame Set the condensor and the luminous field diaphragm for KOHLER illumination With Transmitted Light activated On the halogen lamp is automatically occluded in the laser scanning mode 4 44 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Microscope settings on Axio Imager for reflected light observation Epi fluorescence e Turn on the HBO 100 W power supply with switch 4 29 1 e Click on the RL reflected light button The shutter opens f To avoid excessive bleaching of biological samples expose the specimen to the minimum possible irradiation i e keep the irradiation time as short as possible For this close the field stop to the necessary field of illumination and use additional filters if available e By clicking on the reflector turret button select the
488. tter than 40 nm and the maximum speed amounting to 60 Hz The stage allows the use of specimens with a weight of less than 100 g The piezo stage is not used if manual coarse focusing is performed To position the objective in relation to the optical Z axis the standard XY microscope stage is used The piezo stage features a mount for standard object carriers of 76 mm x 26 mm x 1 mm and a milled out receptacle for 36 mm x 1 mm Petri dishes 7 5 2 Application Fields High precision tine focusing and translation of the object along the optical axis Fast and high precision mounting of one dimensional Z line sections Fast and high precision mounting of two dimensional R Z longitudinal sections Fast and high precision mounting of XY Z Stacks for the three dimensional reconstruction of the object Exact measurement of Point Spread Functions for deconvolution 7 5 3 Additional Information on the Operation The piezo fine focusing stage is a high precision sensitive accessory for the LSM 5 LIVE trom Carl Zeiss and must therefore be treated carefully High mechanical stress such as the use of specimens weighing more than 100 g or the application of pressure or knocks on the movable stage tongue can result in damage and therefore in failure of the stage function To be able to fully utilize the outstanding precision attainable with the fine focusing stage anything which could interfere with its operation especially
489. tton Calculation of the volume parameters Volume 1 Profile measurement mode in 2D display e Select the required 2D display of the stack via the 2D button e Click on the Diagram button in the Measure button bar Click on the Profile button in the button bar displayed afterwards The Table and Profile button bars are displayed below the Measure button bar A colored arrow intersection line of the profile is displayed in the image and the profile diagram appears below the image e f required match the size of the Image Display window in order to obtain the complete display of the profile diagram 4 294 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss ra Frofile Fig 4 295 Topography display 2D Profile Additional Table Profile buttons The additional Table Profile buttons have the following functions g gt g C a j ow qu m aa 4 l m me a 03 06 Show button The profile is displayed in the form of a table at the bottom below of the Image Display window Copy button The profile table is copied to the clipboard and can be transferred to other programs MS Word or MS Excel via the Paste function Save button The profile table can be stored as a text file ASCII Arrow selection button Activation of the mouse button for selection resizing or movement of the intersection line in the imag
490. ttons Note that more formats are available in the format pull down list e Use the Scan Speed slider to adjust the exposure time e Previous the slider is shown the number Frames per Second Start with 2 to 4 frames per second 03 06 OPERATION Acquire Menu TA Scan Control Objective Frame Size Farnak Scan Speed FPS Pixel Depth Scan Direction amp Scan Average Data Depth Method Mean F Scan Direction gt p Co Carl Zeiss Plan Neofluar 100 3 128 256 ole 68 1024 a 512 if 512 512x512 Pixel Time 469 92 usec Scan Time 250 00 msec Zoom Rotation amp Offset Fig 4 50 Scan Control window Mode Frame Objective Lens Image Size amp Line Step Factor Objective Frame Size Format Scan Speed FFS Fig 4 51 B 45 0019 e Flan Negiluar 10 03 F 128 205 512 768 1024 xi s2 vf 2 m Pinel Time 4 Objective Lens Image Size amp Line Step Factor panel 4 71 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan The time slider displays the following information Frames per second it is possible to type in the required FPS directly or to move the slider accordingly Pixel time and Frame scan time Pixel Depth Scan Direction amp Scan Average Pixel Depth Scan Direction amp Scan Average Data Depth 5 Bit 12 Bit geii Frame Ei anel _ Method Mean e Select 8 Bit or 12 Bit Data Depth i e
491. tware and hardware components of the LSM 5 LIVE e In the Main menu toolbar click on Maintain This opens another subordinate toolbar in the Main menu E LSH 5 LIYE File Acquire Process 3D View Macro Options Maintain Window Help as Options Maintain p Reboot HW admin Test Grd Fig 4 218 Maintain menu 3D View aa Acquire T x Process TW Scanner Calibration 4 10 1 Scanner Speed 1 10 elect S Je loptic gt Close The Scanner function is used for scanner calibration Scanner type canne I Display Graphic i The following type of calibrations are available Mean square scanner divergence mas scanner divergence 0 000 Electrical calibration with Speed 1 10 unidirectional bidirectional Progress Status Electrical calibration with Speed 10 11 unidirectional Fig 4 219 Scanner Calibration window 4 10 1 1 Electrical Calibration LS Electrical Calibration with Speed 1 10 can be performed unidirectional bidirectional but with Speed 11 13 only unidirectional 1 Preliminary notes The electrical calibration has to be performed every 2 3 months For electrical calibration no laser scanning is performed and for that reason no calibration sample is needed 4 210 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Maintain Menu Carl Zeiss 2 Calibration conditions Before the calibration process can be started the system has to be in
492. ue those with grey values higher than High are highlighted in red If the Invert option is selected the grey values outside the defined interval will be segmented If the option Binary is selected then all grey values in the range trom Low to High will be set to white grey value 255 in the Output image sequence while all others will be set to black grey value 0 If the option is not selected the grey values within the selected interval remain unchanged while those outside the range will be set to black The measurement function accepts both results without any difference in the results Parameters Input Input image sequence Output Resulting image sequence Colour Green Selected interval is displayed in green Blue Red Grey values below the selected interval are displayed in blue grey values above in red Binary O Selected voxels retain the original grey value 1 Selected voxels are set to grey value 255 the rest to grey value O Invert O Grey values inside the selected interval are segmented 1 Grey values outside the selected interval are segmented L Low grey value threshold C Center of threshold interval H High grey value threshold 6 34 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Segment Automatic The function carries out an automatic grey value segmentation by means of thresholding Interactive Automatic Input ho Output 2 Hew Colour Green C Blue Red
493. ue range Input Input H Upper boundary of grey value range Input Output L Lower boundary of grey value range Output Output C Center of grey value range Output Output H Upper boundary of grey value range Output Smooth Gauss This function performs a Gauss filter rou B aree Dutput Moo New Size Bo HE H 3 31 Cancel Apply Fig 6 16 The noise in the image sequence is reduced the edge shape is nearly unchanged local maxima are leveled the dynamic range is reduced Image sequences should be smoothed before they are reconstructed or segmented For most sequences a Size value of 3 Is sufficient enough If Input is a multichannel sequence any number and combination of channels can be selected Output will only get the selected channels as results The grey value of every pixel is substituted by a weighted average of its surrounding neighbors The neighbors are defined by a cube The affected pixel is the central pixel of the filter cube The weighted filter cube is approximated by a binomial distribution The size of the filter cube Is set using the Size scroll bar Even numbers are set to the next odd value The Size defines the strength of the smoothing Parameters Input Input image sequence Output Output Image sequence Size Filter size 3 31 only odd numbers 6 24 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Morphology The following four functions perform basic operations
494. uence and also if the objective is to make deeply layered structures visible 6 46 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Method The Input sequence defines the data to be reconstructed If it is a multichannel sequence one or all channels can be selected for the reconstruction Output sets the name of the result image sequence If the sequence exists it is overwritten Pressing the button New will generate a new name number The size of the sequential images in Output is determined by the size of the sequential images in Input Number of Views determines the number of reconstructions which should be computed The radio buttons Start and End define which angle settings are currently shown A definition for the angle End is only necessary if Number of Views is higher than 1 If this is true the result sequence will get views from the Start to the End angle definition The other reconstructions are determined through the linearly interpolated intermediate angles The direction of view is determined from the angles as follows The angle Angle Z determines the rotation of the direction of view on the Z axis The angle Angle Y determines the rotation of the direction of view on the Y axis that has been rotated by the angle Angle Z The angle Angle X determines the rotation of the direction of view on an X axis that is rotated by Angle Z and Angle Y Channel defines if the following parameters a
495. ues with the arrow keys click once into the histogram Using the left or right arrow key by its own will move the whole range Pressing the Shift key additionally moves the lower boundary the Control key the upper boundary The vertical scale of the histogram is set using the scroll bar The units are percents of the maximum grey value distribution This setting has no influence on the function Parameters Input Input image sequence Output Output image sequence Channel Selection of the channel numbers for the Output image after contrast enhancement Clip Grey Values Clipping of grey values to the Low L and High H output grey values boundaries Input L Lower boundary of grey value range Input Input C Center of grey value range Input Input H Upper boundary of grey value range Input Output L Lower boundary of grey value range Output Output C Center of grey value range Output Output H Upper boundary of grey value range Output 6 20 B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Contrast Automatic This function scales the grey values of an image sequence to the maximum possible range 4 Contrast E4 Interactive Automatic Linearize l Input Moo ma Dutput 20 i efe Channel Alf 1 Threshold lo 4 r O 1000 Input Histograrn L fo ih E 127 pl H 255 KG 0 255 ah Outout Histogram Ao RH Cfi RN Hss RN 0 255 OK Cancel Fig 6 14 The Automat
496. uire subordinate toolbar of the Main menu The Time Series Control window appears on the screen e Click on the Close button to close the Time Series Control window The following functions are available on the right hand side of the Time Series Control window Close button Closes the Time Series Control window New button Opens a new Image Display window Start T button Starts the Time Series Stop button Stops the entire Time Series A current scan is interrupted Pause button Interrupts the Time Series Button labeling is changed to Resume A current scan is performed until the end When the button is pressed again the Time Series is immediately continued with the next scan procedure 4 94 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss Mean ROI button Creates a Time Series with the intensity values of the Frame or the default ROIs An average value is formed of the intensity values of the Frame or the ROls determined These average values are displayed in an extended Image Display window as a function of the time which has passed The status line in which the phases of the current Time Series or notes for the user are displayed is in the lower part of the Time Series Control window 4 5 6 2 Start Series Panel Tri Ti In this panel the parameters for the start of the li Time time series are set Trigger out None Fig 4 79 Start Series panel The following function
497. under warranty including parts not directly affected by such action This also includes the modification of the system computer with new cards etc by the user The use of a camera at the base port of Axiovert 200 M Combi stands is not allowed for reasons of laser safety Any manipulation will result in the loss of warranty of laser safety Please read also the notes on device safety and manuals of the microscope the HBO the HAL and additional optional devices if ordered as the UV Laser the piezo focusing device and heating inserts As the system is largely operated via menus on a computer you should be acquainted with the principles of the operating system and its WINDOWS WINDOWS 2000 or Windows XP graphical user interface The respective manuals are supplied together with the programs Ay The LSM 5 LIVE is a device that belongs to laser hazard class 3B The system is equipped with safety interlocks that comply with laser hazard class 3B and 4 WHO recommendations concerning health and industrial protection when handling laser devices must be observed The operator of the unit must also observe all and any relevant statutory accident prevention regulations The user is referred to the safety data sheet provided together with the manual 1 2 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Warning and Information Labels Carl Zeiss 1 2 Warning and Information Labels A The warning and information labels attached o
498. uous zooming of the 3D reconstructed image A click on the Any View button displays the 3D reconstruction image in a shadow projection where the viewing point can be defined In addition to the zoom setting the image can be rotated around the three orthogonal axes via the relevant setting wheels However the 3D orientation can also be set directly in the Image Display window by clicking holding and dragging the 3D reconstructed image with the mouse Convallaria AIM Display x Transparency Basic Advanced Surface Basic Advanced Full Res Appearance Sees gt MC Ready 51285128 20 3 channels 3 bit Fig 4 273 Image Display window 3D display Shadow projection Any View The following additional buttons are available in the Any View shadow projection mode e After activation of the Frame button below the image a bounding box is drawn around the 3D reconstructed image 3 Depending on the used mode and hardware configuration it can take several seconds until the 3D reconstruction is refreshed on the monitor after reorientation e A click on the Coordinate System button displays a colored coordinate system in the Image Display window where the X axis is displayed in red color the Y axis in blue and the Z axis in green e A click on the Scale button display an X Y and Z scale in the Image Display window e Aclick on the Home button resets the display param
499. urrent image under a new name Imports images Exports Images The current image is displayed on the full screen Deactivation of the function with a click of the mouse Several images are printed on one page Use of the RAM memory for image display Use of the hard disk as storage medium for image display The Zeiss LSM Image Browser main menu is closed The functions New Open Save Save As Import Export and Multi Print correspond to those of the Expert Mode of the LSM 5 LIVE software and have already been described in chapter 4 03 06 B 45 0019 e 5 5 TOOLS LSM 5 LIVE Carl Zeiss LSM Image Examiner LSM 5 LIVE Duo Scan 5 4 LSM Image Examiner The LSM Image Examiner can be used without having to open the LSM 5 LIVE software However this requires the installation of the relevant dongle The LSM Image Examiner provides all the functions of the LSM Image Browser plus the 3D functions and selected Process functions of the Expert Mode of the LSM 5 LIVE When images are opened a large scope of the image processing functions of the LSM 5 LIVE software is available for further details see chapter 4 e Click on the LSM Image Examiner icon on the desktop of the PC The Zeiss LSM Image Examiner main menu is opened i Zeiss L5H Image Examiner File View Process Options Window Help CHE E Duplicate Contrazt Fig 5 7 Zeiss LSM Image Examiner main menu In addition to the buttons of the LSM Image Browser mentioned above t
500. us of all Diode 40850 408m lasers available DPSS 532 75 Baz nm The subordinate laser settings panel shows the relevant and currently set Maximum Power J Diode 485 100 Wavelength Status and Tube Current only posmam Forat E POU Uee Sapphire 488 of the current laser wavelength 488 nm Bete Beady a The buttons On Off and Standby permit the current laser to be set in the required status The name of the selected laser Toptica etc is displayed in the headline of this setting panel for Fig 4 25 Laser Control window checking 4 5 1 1 Switching on the Enterprise UV Laser e f the UV laser is required switch it on via the toggle switch 4 26 1 of the power supply It will be ready for operation after a few seconds Fig 4 26 Power supply of UV laser 4 5 1 2 Opening Closing the Laser Control window e Click on the Laser button in the Acquire subordinate toolbar This opens the Laser Control window which shows all lasers connected to the system When the setting of the required lasers has been finished the Laser Control window can be closed again e Click on the Close button to close the Laser Control window The Laser Control window will be closed 4 38 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss 4 5 1 3 Function Description Lasers panel upper List of available lasers including the display of relevant wavelengths and Switching status
501. utomatically linearly interpolated between the initial and end values see page 4 84 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Acquire Menu Carl Zeiss The parameters of a Z Stack can be defined using the Z Sectioning tab the Mark First Last tab or if the optional Piezo focus for objectives is connected the Hyperfine Z Sectioning tab Z Sectioning tab Num Slices Interval Current Slice Keep Interval Keep Slices 03 06 Entry of the number of sections single XY images to be recorded with the stack via the slider arrow buttons The entry does not influence the interval Entry of the step width Z distance between the single XY images via slider arrow buttons The entry has no influence on Num Slices Display of the current position of the slice within the stack Change of position via slider arrow keys Reset of the current slice position in the center of the stack by clicking on the C button Of course the borders of the stack are also changed if the current slice position is changed The interval remains constant when the stack limits or number of slices are changed The number of slices remains constant when the stack limits or interval are changed i The Stack Z Size is slightly adjusted to hold the number of slices with no interval changes or the interval size if the number of slices is varied Mark First Last Hopere 2 Sectioning Mum Slices 20 4
502. utton bar the Line Dist and Line Offset sliders input boxes are displayed e Select the required Iso Line display mode by clicking the left mouse button 4 284 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss The additional function elements of the Iso Lines display mode have the following meaning Line Dist Line Dist slider Changes the distance of the iso lines Line Offset Line Offset slider Setting of the height level where the Iso Lines display starts f To apply the topography functions to a small portion of the Z Stack image use the Overlay function Overlay button and cut out and store as new topographic evaluation via the Extract Region function 3 3D display Topo animations are possible The following 3D modes can be set Profiles button Protile display Profiles Grid button Grid display Filled button Display of color shades Shaded button Surface rendering Can be combined with LUT Topo animations are chaded possible tE ee A e Click on the 3D button in the Display button bar The 3D display mode selected last is activated At the same time an additional button bar appears beside the 3D button to permit selection of the required 3D display mode Below the Measure button bar the Scaling button bar and the Profile Dist and Fill Level Sliders input boxes are displayed The Image Display box contains one horizontal and
503. vailable Macro StitchArt plus 4 328 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Software and Hardware Options Carl Zeiss 4 16 2 Hardware Depending on whether the following hardware components are available or not the content of the screens may differ Piezo objective focusing device X Y scanning stage DC 4 x 4 or DC 100 x 90 each with MCU 28 Stands Axio Imager Z1 Axiovert 200 M Depending on the configuration the scan head equipment may differ in filters beam splitters and the number of photomultiplier Transmitted light PMT Monitor diode If your configuration does not include the Piezo objective focusing device the following functions are not available Hyperfine Z Sectioning in the Z Stack function in the Scan Control window HRZ parameters in the Stage and Focus Control window If your configuration does not include the X Y scanning stages DC 4 x 4 or DC 100 x 90 each with MCU 28 the following functions are not available Stage Position and Tile Scan functions in the Stage and Focus Control window Depending on the used microscope stand Axio Imager Z1 or Axiovert 200 M the following dialogue and available functions may differ Context and accessibility of the Microscope Control window If your configuration does not include an AxioCam the following functions are not available Camera in the Config Control window Scan Control window 03 06 B
504. value to enable a confocal fluorescence XY image to be obtained A small aperture size will increase the depth of focus but reduce the light intensity received by the CCD detector The influence of the aperture size on image creation is shown by the example in Fig 4 310 The entire image was first scanned with too large a aperture size The aperture size was then optimized for a defined ROI This considerably improved the display of the specimen structures e Click on the Palette button in the Select image processing toolbar This opens the Color Palette window e In the Color Palette List panel click on the Range Indicator item The scanned image appears in a false color presentation OPERATION Image Optimization Carl Zeiss Female TOGA x TOG Z chiare g b Fa pepe data Dpi Zoom 1 _ Pike Abo Paita Fig 4 310 Image Display window with confocal ROI Color Palette Ea Color Palette List Close Add to List Import ki o m C Green Humber of 7 C Blue knots Reset i All Color Palette window Fig 4 311 03 06 B 45 0019 e 4 319 OPERATION LSM 5 LIVE Carl Zeiss Image Optimization LSM 5 LIVE DuoScan If the image is too bright it appears red on the screen ia m Pa Tei aah ea Feri TOE x IZE Z chire g bi Fa pepe date Depi ea E Fale Abo Palaia Fig 4 312 Image Display window If the image is not bright enough it appears blue
505. ve the XY image Section of the YZ plane red line through the stack right of the XY image Section of the XY plane blue slice plane of the stack center image 03 06 B 45 0019 e 4 247 OPERATION LSM 5 LIVE Carl Zeiss Display and Analysis of Images LSM 5 LIVE DuoScan 4 13 17 2 Ortho Distance Function e Activating the Dist button permits length measurements in 3D space e Click on the Mark button to set the first XYZ point for the measurement of the spatial distance e Set the second XYZ point for measurement by moving the X Y Z sliders or by moving the green red and blue lines in the image The projections of the spatial distance are shown in the image planes by yellow lines The actual Spatial distance is calculated and shown in um below the Select Dist and Mark buttons e g 3D Distance 55 60 um convallaria AIM Yellow line Ready S12 2 512266 3 channels 8 ba Fig 4 255 Image Display window Ortho Distance display 4 248 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss 4 13 17 3 Ortho 2D DeConVolution Function The 2D deconvolution function causes orthogonal projection enhancement through the computed correction of the resolution in the Z coordinate Image enhancement Is only effective for the two projections of a fluorescence stack in the Ortho display or fo
506. vert 200 M stand is moved to the load position prior to switching off the LSM 5 LIVE system and the piezo stage is then moved to the lowest position to avoid damage of the objective or object by a possible collision The user must refocus after start up of the system Before an objective change in the Axiovert 200 M or the Axio Imager Z1 the nosepiece and the microscope stage must be moved to the load position by the user and then back to the work position to prevent the objectives from hitting the piezo components This is performed automatically if the objectives are changed menu controlled via the relevant buttons of the LSM 5 L VE program 7 8 B 45 0019 e 03 06 LSM 5 LIVE ANNEX LSM 5 LIVE DuoScan Piezo Objective Focussing Device Carl Zeiss 7 6 Piezo Objective Focussing Device For upright stands Axio Imager Z1 Axio Imager M1 Axioskop 2 FS MOT Range 250 um Minimum step size 5 nm Piezo objective f Piezo focussing stage ocussing device Step size lum Objectives WO0 8 M27 Modified Achroplan 40x 0 8 W with reduced length to compensate for piezo height Installation e Screw in your microscope objective into Piezo Objective Focusing Device see Fig 7 2 1 e Screw the thread ring into your microscope see Fig 7 2 2 e Easy clamp the Piezo Objective Focusing Device on the thread ring see Fig 7 2 3 Fig 7 2 Installation of the Piezo Objective Focusing Device 03 06 B 45 0019 e 7 9 ANNEX
507. w B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Render Alpha Method Description Each Output pixel is a weighted sum of the Input voxels along a ray in view direction through the Input sequence Each Input voxel has an opacity value dependent only on its grey value The opacity values are defined by the parameters Threshold Ramp and Max Opacity Accumulation of pixels proceeds along the ray trom back to front i e from far pixels to near pixels If a new pixel is added it increases the result intensity by its grey value weighted by the opacity value and attenuates the previously accumulated intensity according to the opacity value Full intensity stops accumulation This calculation must be repeated for each pixel of the ray to generate one Output pixel Then for each Output pixel to produce a 2D Output image for the selected view angle Then for each view angle to produce an output sequence for Number of Views different view angles Render References 1 J D Foley A van Dam S K Feiner J F Hughes Computer Graphics Principles and Practice Addison Wesley Reading MA 1990 2 M Levoy Display of Surfaces trom Volume Data IEEE Computer Graphics amp Applications May 1988 29 37 3 J Yla Jadski F Klein O Kubler Fast Direct Display of Volume Data for Medical Diagnosis VGIP Graphical Models and Image Processing 53 1991 7 18 4 K H H hne R Bernstein Shading 3D Images f
508. will be performed e Use the focusing drive of the microscope to focus on the upper position of the specimen area where the Z Stack is to start e Click on the Mark First button to set the upper position of the Z Stack e Then focus on the lower specimen area where the recording of the Z Stack is to end e Click on the Mark Last button to set this lower position e Click on X Z 1 1 1 button to set the Z interval in such a way that the voxel has identical dimensions in the X Y and Z directions cube e Click on the Start button to start the recording of the Z Stack 03 06 Storing an image e Click on the Save or Save As button in the Image window or in the File subordinate toolbar of the Main menu The Save Image and Parameter As window appears Save Image and Parameter As 1 Channel Flucrescene D VAIMAGUIDED Guided mdb Q Example Image Databases Biohed Biokd ed mdb QI Example Image Databases M ateral aterial mdb Qi Example Image Databases NWLONLO mdb Fig 22 Save Image and Parameter As window e Enter file name description and notes in the appropriate text boxes e Click on the OK button Switching off the system e Click on the File button in the Main menu and then click on the Exit button to leave LSM 5 software program Fig 5 e Shut down the computer e Put the LASER key switch to OFF position Remove the key from the system rack e Put the COMPONENTS switch and the Sys
509. window of Channels 1 and 2 of the printed manual 4 Beam path Activation Deactivation of Channel en Sa ea Channels and Channel Color Assignment 3 Coor M E el ml e On the Beam Path and Channel Assignment Close panel click on the channel symbols e g ical This opens the Channel Color Selection window on the Beam Path and Channel Define Assignment panel e Click on the desired color bar Fig 4 40 Channel Color Selection window This changes the color of the channel symbol e To close the Channel Color Selection box click on the Close button 03 06 B 45 0019 e 4 57 OPERATION Acquire Menu Carl Zeiss a Reticule Hue ise a 213 bun iis Reg 222 Green 15 Biye 233 et Channel Colors window Fig 4 41 ISJ LSM 5 LIVE LSM 5 LIVE DuoScan Further colors for the corresponding channel can be produced as follows Clicking on the Define button will open a further Channel Colors window All the available colors are shown as buttons in the Current Set of Channel Colors panel Via a reticule in the Define Color panel any desired color can be produced Clicking on the Add button allows the color to be used for further channel coloring Choose the desired color with the reticule the reticule is in the left corner at the bottom of the color range Define the brightness by use of the scroll bar Use the Add button to add the color to the colo
510. wing shapes numbered 1 to 3 from left to right are available If Grey Morphology is selected the function will respect all grey value shades of the sequence Input If Grey Morphology is not selected the function will distinguish between O and non O only The result Output will be a binary sequence Parameters Input Input image sequence Output Resulting image sequence Shape Shape used 1 Cross 2 cube 3 cube cross Count Number of recursive operations 6 30 Grey Morphology 0 Distinguish between 0 and non O only 1 All grey value shades are taken into account B 45 0019 e 03 06 LSM 5 LIVE 3D FOR LSM LSM 5 LIVE DuoScan Functions Carl Zeiss Morphology Open This function carries out an opening HH Horphology Erode Dilate Open Close Input i all 1 Output Booo Hem Shape Be Count Poo w 11 0K Cancel W Grey Morphology Fig 6 22 In the Morphology dialog window the tab sheet Open must be selected This function carries out an erosion followed by a dilation For the most part the opening maintains the Original size of the regions Thin connections between regions and small regions themselves disappear Convex bulges in the contours of the regions are reduced The opening is applied to the grey value image sequence Input Count times with the shape Shape If Input is a multichannel sequence any number and combination of channels can be selected Output will onl
511. wing tools in the Mean ROI image display If a region has been selected a cross appears next to the button Opens a further dialogue with the following options Add Region New Group Remove Group and Remove All It provides the possibility to chose more than one ROI for analysis and also to group them together according to the experimental set up First click Add Region than click into the ROI that has been drawn using the drawing tools in the Mean ROI image display For each added ROI a cross is displayed in the line Click Add Region for each ROI to be added Clicking New Group puts the cursor in the next line and further ROIs or Group of ROIs can be added for analysis Performs the calculations and the fitting of the data B 45 0019 e 4 147 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 17 3 Example FRAP Performed in a Nucleus Expressing GFP Labeled Proteins Display of the image series in the Mean ROI display mode The drawing tools are used to define the ROI to be analyzed ROI 1 the background ROI ROI 2 and the reference ROI ROI 3 The reference ROI must be a neighboring cell which has been imaged with the same laser intensity over time identical to the cell which has been bleached to induce FRAP Make sure the whole cell or cell compartment of interest Is imaged and therefore illuminated gt Use the Slice submenu in the Select toolbar of the image window to display the image right after the bleach eve
512. xel Shitt 544 do gt gt possible size which produces a good high Postion 54 Ao gt gt contrast image This setting changes the aperture size The ZSlice display box simultaneously Store Current a Move To Stored a displays the depth resolution corresponding to the aperture size yape menl e eoe EA i Image optimization can be effected with I Fast Adjust Mode the Range Indicator or in the Line Scan mode Fig 4 230 Pinhole panel e Optimize the pixel shift and the aperture position in Y relative to the PMT using the Pixel Shift and Position Y sliders to maximum image brightness e Click on the Save Current Position button to save the aperture adjustment e Removing the Position slider in the Collimator panel allows the collimator to be adjusted to maximum image brightness Optimum collimator adjustment obtained in this way can be stored by clicking on the Save Current Position button e Click on the Stop button to stop the continuous scan RS Please do not make any program manipulations while the automatic aperture adjustment is running status display is red busy 4 218 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 2 Automatic aperture and collimator adjustment The automatic adjustment allows the LSM 5 LIVE apertures to be used with any combination of beam splitters e Click on the Adjust Automatically button The Requirements for Adjustment window will then
513. xioCam MRm Axioskop 2 FS mot 2 14 B 45 0019 e LSM 5 LIVE LSM 5 LIVE DuoScan Illuminator HBO 100 with lamp mount and collector HBO 100 illuminator self adjusting with lamp mount and collector Power supply unit for HBO 100 FluoArc variable intensity lamp control for HBO 100 X Cite 120 fiber coupled illuminator HAL 100 illuminator with collector Halogen lamp 12 V 100 W HAL 100 illuminator with collector Halogen lamp 12 V 100 W Power supply 12 V DC 100 W stabilized 03 06 LSM 5 LIVE LSM 5 LIVE SETUP REQUIREMENTS LSM 5 LIVE DuoScan System Overview LSM 5 LIVE Material Carl Zeiss 2 18 System Overview LSM 5 LIVE DuoScan 3 AxioCam HRm AxioCam HRc AxioCam MRm X Cite 120 fiber coupled illuminator Microscope stand Axio Imager DUO with TFT monitor and light control mot HAL 100 illuminator with collector Halogen lamp 12 V 100 W gz NELA Switching A mirror mot AxioCam HRm AxioCam HRc AxioCam MRm S vad SX L Axiovert 200 M DUO Several solutions for incubation are available AxioCam HRm AxioCam HRc AxioCam MRm i S Transmitted light channel for LSM 5 HAL 100 illuminator with collector Halogen lamp 12 V 100 W Power supply 12V DC 100 W stabilized REGLA Switching A mirror mot Transmitted light Axioskop 2 FS mot DUO channel for LSM 5 03 06 B 45 0019 e 2 15 LSM 5 LIVE INTRODUCTION TO LASER SCANNING MICROSCOPY LSM 5 L
514. xxxx Baujahr 20xx ECU LSM510 LIVE 000000 1291 810 3 N PE 400 230V AC 50 60 Hz max 5000VA CE Carl Zeiss CE Ser Nr 2420Xxxxxx ECU LSM510 LIVE 000000 1291 810 AN o il I afi Carl Zeiss ae LASER RADIATION AVOID DIRECT EXPOSURE TO BEAM Carl Zeiss Jena GmbH 07740 Jena GERMANY 488 nm 100 mW Ser Nr XXXXXXXXXX 532 nm 75 mW Lasermodul RT M 1300 608 440 nm 20 mW 1 N 240 120V AC Laser module LSM 5 LIVE 405 nm 50 mW 50 60 Hz 600 VA 635 nm 40 mw C CLASS IIIb LASER PRODUCT Fig 1 4 Warning and information labels on laser components 1 8 B 45 0019 e 03 06 LSM 5 LIVE NOTES ON DEVICE SAFETY LSM 5 LIVE DuoScan Warning and Information Labels Carl Zeiss DANGEROUS VOLTAGES DANGEROUS VOLTAGES UNDER THIS COVER UNDER THIS COVER l LLLLZZZZ LLLILLLIJ v v DANGEROUS VOLTAGES UNDER THIS COVER VISIBLE AND R INVISIBLE LASER RADIATION AVO l D EX POS U R E IS EMITTED FROM THIS APERTURE Laser Enterprise II 653 80 mW 351 nm 364 nm VISIBLE AND INVISIBLE LASER RADIATION UV Laser module AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION ARGON ION LASER 2 WATTS MAX CW PRACTICAL LIMIT CLASS IV LASER PRODUCT Fig 1 5 Warning and information labels on UV laser components LSM DuoScan only 03 06 B 45 0019 e 1 9 NOTES ON DEVICE SAFETY LSM 5 LIVE Carl Zeiss Regulations LSM 5 LIVE DuoScan 1 3 Regulations Extensive knowledge of the hardware the system is indispensable for safe
515. y To remove a condition line double click on it The parameter Minimum Volume defines the minimum voxel volume for the measurement This is an easy way to eliminate very small regions caused by noisy sequences and segmentation process The button Remove All will clear the list of defined conditions Clicking on the Apply button will execute the measurement process and switch to the General tab sheet of the dialog Parameters Feature List of available object features Operator List of available condition operators Number List of numbers to compose the value List of conditions Defined condition list Remove All Remove all entries trom the List of conditions Minimum Volume Minimum object volume in voxel 03 06 B 45 0019 e 6 5 7 3D FOR LSM LSM 5 LIVE Carl Zeiss Functions LSM 5 LIVE DuoScan Automatic Object Measurement General This function carries out an automatic measurement and labeling General Object Features Volume Features Condition Mask Image ze OoOO i Dens Image Moo i Output Object Database objet I Clipboard Volume M Database volume Label cd Image flate New Object Visualisation M d easure Image i H Render m obiect New Object Features No Volume Surbarea MeanD Std Surkarear micrometer 3 micrometer 2 grey grey micrometer 2 3937 50 3550 00 3550 00 1187 50 975 00 975 00 i 687 50 825 00 825 00 P 2000 00 1387 50 1387 50 Ss 2125 00 1587 50 1587 50 VolCount
516. y changing the values of the listed properties Using this block enables to copy one image into another with the coordinates and settings put into the properties list The destination image is connected to the left input the source image Is connected to the right input The subsequently arriving images will be concatenated according to the coordinate setting and treated as one image A subsequent image display will be updated each time a new image arrives The second concatenate action block allows to concatenate images from two different acquisition blocks B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Macro Menu Carl Zeiss Hardware Control Action Blocks If a motorized stage is available the movement of the stage can be set up as ee part of the program flow The stage can be moved to an absolute position 5 which can also be taken from the actual stage position using the Read back function or typed in individually Using a relative position will use the current stage position as relating position The values have to be typed in F The movement of the focus can be set up as part of the program flow The Ht pe focus can be moved to an absolute position which can also be taken from the actual focus position using the Read back function or typed in individually Using a relative position will use the current focus position as relating position The values have to be typed in For large travel ranges which exceed half of th
517. y difference is that the mathematical formula is based on subtraction 4 124 B 45 0019 e 03 06 LSM 5 LIVE LSM 5 LIVE DuoScan 4 6 3 Multiply The Multiply function permits two channels each to be linked into a new channel by multiplication The channel created in this way can be stored via the Save As function 4 6 3 1 Open Close the Multiply Window e Click on the Multiply button in the Process subordinate toolbar of the Main menu This opens the Multiply window e Click on the Close button to quit the Multiply window 4 6 3 2 Performance of the Multiply Function This function is performed in the same way as the Add function see Add page 4 121 The only difference is that the mathematical formula is based on multiplication OPERATION Process Menu Carl Zeiss EG Multiply Close Click into window Apply Channel Chi v Destination New Image 12 bit Channel New Source 1 10 00 Source2 10 00 64 00 Vv Preview ja 8 00 Fig 4 113 Multiply window 03 06 B 45 0019 e 4 125 OPERATION LSM 5 LIVE Carl Zeiss Process Menu LSM 5 LIVE DuoScan 4 6 4 Ratio The Ratio function permits to create a new image or image series by offsetting two images or image series against each other The channel created in this way can be stored via the Save As function 4 6 4 1 Open Close the Ratio Window e Click on the Ratio button in the Process C
518. y get the selected channels as results The Count scroll bar determines the number of recursive operations The following shapes numbered 1 to 3 from left to right are available a If Grey Morphology is selected the function will respect all grey value shades of the sequence Input If Grey Morphology is not selected the function will distinguish between O and non O only The result Output will be a binary sequence Parameters Input Input image sequence Output Resulting image sequence Shape Shape used 1 Cross 2 cube 3 cube cross Count Number of recursive operations Grey Morphology 0 Distinguish between 0 and non O only 1 All grey value shades are taken into account 03 06 B 45 0019 e 6 3 1 Carl Zeiss 3D FOR LSM LSM 5 LIVE Functions LSM 5 LIVE DuoScan Morphology Close This function carries out a closing HH Horphology Erode Dilate Open Close Input i All 1 Output 2 New Shape Ba Ee Count Poo w ppt 0K Cancel IY Grey Morphology Fig 6 23 In the Morphology dialog window the tab sheet Close must be selected This function carries out a dilation followed by an erosion For the most part the closing maintains the Original size of the regions Connections are formed between adjacent regions gaps and bright concave bulges in the contours of regions are Tilled in The closing is applied Count times to the grey value image sequence Input with the sha
519. y where you want the text file to be stored enter a file name and click on Save A text file containing the topography in the form of an XYZ matrix Is generated 3 Copy profiles to clipboard item OPERATION Display and Analysis of Images Carl Zeiss Metric egual ratio woe T and scaling s and scaling No scaling Esport profiles Copy profiles to clipboard Export sz triples Copy z triples to clipboard Show processing parameters Ratio of Valid Data Points Load Calculation Parameters Save Calculation Parameters Render Series Fig 4 284 Context menu of the 3D display mode Profiles Save As Save in Lem510 amp El c ls cee 3 helptiles LOM 510 Rel 2 02 L Ein CI Hwt I Database C Images D bfilter CI Macros Rois LemS10_2 3 A LSMIB_AEADM 2 LSMix_READM ttt tt File name Jtestl tet Save as type Text Files txt Cancel Save As window Fig 4 285 x 000 2 10 55 2 21 10 2 51 65 2 42 19 Yio232 0 00 0 00 0 00 0 00 0 00 Y 12 87 0 00 5 71 T A T02 3 99 Y 23 42 0 00 6 62 6 96 TAD 5 77 Y 33 97 0 00 3 02 7 82 7 99 6 11 Y 4441 0 00 5 94 7 76 731 5 63 Fig 4 286 Topography matrix All 3D parameters shown right from the main 3D topo display window are copied in the clipboard 03 06 B 45 0019 e 4 287 Carl Zeiss Save As Save in J Bin Dbfil Filter Database H vut ter 9 Images C Macros C Rois exp prot tut
520. yed separately for each ROI used Mono button Switches between color and monochromic display of intensity profiles Automatic button Automatic scaling of the display of Intensity Time diagrams Time Range button Display of Intensity Time diagrams is scaled depending on the Time Range set in the input box shown on the left Number Cycles button Display of Intensity Time diagrams is scaled depending on the Number Cycle set in the input box shown on the left Show Image button Shows the scan image in the Image Display window to the side of the intensity diagram This button is active only If the Live Image option is activated Copy Table button The table of intensity values is copied to the clipboard Show Table button The table of intensity values is displayed at the bottom left of the Image Display window Save Table button The table of intensity values can be stored as a text Tile B 45 0019 e 4 107 OPERATION LSM 5 LIVE Carl Zeiss Acquire Menu LSM 5 LIVE DuoScan 4 5 7 Stripe bleach function LSM 5 LIVE without LSM DuoScan Bleaching of selectable rectanqular image regions with the LSM 5 LIVE Stripe Bleach is activated by the EditBleach button in the main menu The Bleach Control window opens and can be used in the same way as in any other Zeiss LSM 5 e g the LSM 510 The only difference is that the bleach region has to be rectangular i LS 5 LIVE Fie Agee Poe Mive Meo Opia Marten Wiel i a m t F owe f uo Ja A
521. zoomed by various methods The zoom function can be performed online e Click on the Zoom button in the Select toolbar The Zoom toolbar will be displayed on the right hand side of the Image Display window e Clicking on the Zoom button again will remove the Zoom toolbar rg i convallana Alh Display cca Select E Chan Ready 1024 x 1024 a channels 12 bit Reconstruction Display Zoom 1 Palette No Palette Fig 4 240 Image Display window Select Zoom Zoom Auto The image is fitted automatically to size of the Image Display window Zoom Resize Restores the image to its initial size Zoom Enlarges the image by factor 2 Zoom Reduces the image by factor 2 Zoom 1 1 Restores an image zoomed in any way to its original size Zoom Mouse Allows you to enlarge reduce the zoom factor of an image using the left right mouse button provided that the cursor is inside the image 4 228 B 45 0019 e 03 06 LSM 5 LIVE OPERATION LSM 5 LIVE DuoScan Display and Analysis of Images Carl Zeiss All When active it allows you to zoom all images of a gallery to the same extent without changing the display image format i Zoom Zoom Zoom 1 1 Zoom Mouse and All can only be defined when the Zoom Auto function is deactivated Slider with display box The zoom factor can be set by moving the slider The display box below displays the current zoom factor Factor 1 corresponds to the original size 4 13 5 Sel

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