Zen 2009 Quick Start Guide
Contents
1. ger When a multi dimensional acquisition tool is not selected the respective tool and its set parameters are not included in the multidimensional image acquisition If no multidimensional tool is activated the Start Experiment button is grayed out and only single images can be scanned 10 2009 19 Storing and exporting image data Fig 25 Save Image buttons in ZEN Se ll hss Era l ahah ehr Fao A 5 ve bb o LN Sf trai Cancel a Fig 26 Save as window Tapp maga Pie Comms of Pega doy m ple Coes y ge ETE E n BPE siaii e agbs peat Fui PEE 6 Sy rE Fig 27 Export window 20 e To save your acquired or processed images click on the Save or Save As button in File Menu button in the Main Toolbar Fig 25 1 or click on the EJ button at the bottom of the File Handling Area Fig 25 2 or click the e The WINDOWS Save As window appears e Enter a file name and choose the appropriate image format Note the LSM 5 format is the native Carl Zeiss LSM image data format and contains all available extra information and hardware settings of your experiment e Click on the SAVE button If you close an image which has not been saved a pop up window will ask you if you want to save it Choosing yes will lead you to the WINDOWS Save As window To export image display data a single optical section in raw data format or the contents of the image display window including analysis and2. o Microscopy from Carl Zeiss LSM 5 MP LSM 510 and LSM 510 META Laser Scanning Microscopes LSM Software ZEN 2009 October 2009 We make it visible Page CONTENTS sists ate nec mmc A cece eo ee E E AE A 1 M OCU CUNO ING ses occ sestincs A E E a siaccuneeuun treoueedseumaecnduns 1 Starting the SYSTE igs nnana sb aera tle aE aana E twee iE Eana 2 Introduction to ZEN Efficient Navigation c ccccceeceeseeseeeeeeeeeseeeeeeeseeaseeeesseeeneeeesseoaaaaes 5 Setting up the MICrOSCOpe s s ssnnssnnnnsnnnnrnnnnrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn ennn mnnn n nennen 10 Configuring the beam path and lasers cccceseeseeeeeeseeeseeeeeeeeeseseeeeseeaaeeeeseeaesseeseeaaaaaes 12 Scanning ah IMAGE esnie E E ia 15 Storing and exporting image data ccccsecceeeeseeeeeeeeeeeeeeeeneeeeeeaneeseeeeseesseeneseeaeneeeseoeneeess 20 Switching off the SVS COUN ean a cineca 21 Introduction This LSM 510 LSM 510 META LSM 510 NLO Quick Guide describes the basic operation of the LSM 510 LSM 510 META LSM 510 NLO Laser Scanning microscope with the ZEN 2009 software The purpose of this document is to guide the user to get started with the system as quick as possible in order to obtain some first images from his samples This Quick Guide does NOT replace the detailed information available in the full user manual or in the manual of the respective microscopes Axio Imager Axio Observer Axioskop 2 FS MOT
3. Also this Quick Guide is written for a user who is familiar with the basics of Laser Scanning Microscopy For your safety Observe the following instructions The LSM 510 LSM 510 META LSM 510 NLO laser scanning microscope including 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 X Cite 120 lamp HBO 100 mercury lamp 10 2009 1 Starting the System Switching on the LSM system e When set to ON the REMOTE CONTROL switch labeled System PC provides power to the computer This allows use of the computer and ZEN software offline Fig 1 e To completely switch on the system now press the Components switch to ON This starts the other components and the complete system is ready to be initialized by the ZEN software Switching on the X Cite 120 or the HBO 100 mercury lamp Fig 1 REMOTE CONTROL switch e Switch on the main switch of the X Cite 120 HBO 100 lamp for reflected light illumination via the power supply as described in the respective operating manual Switching on the Enterprise UV Ar Laser e f the UV laser is required switch it on via the toggle switch Fig 2 1 on the powe
4. list box AA A click on this arrow button will move the selected track highlighted in light grey one position downwards in the list box 14 10 2009 Scanning an image Setting the parameters for scanning e Select the Acquisition Mode tool from the Left Tool Area Fig 18 e Select the Frame Size as predefined number of pixels or enter your own values e g 300 x 600 in the Acquisition Mode tool Click on the Optimal button for calculation of appropriate number of pixels depending on objective N A and x The number of pixels influences the image resolution Acquisition Mode Objective EC Plan Neofluar 100 3 Scan Mode Frame T Frame Size X 128 Y 128 Optimal speed Pixel Dwell 640 psec Scan Time 491 52 msec Averaging Number Bit Depth 6 Bit Scan Area Fig 18 Acquisition Mode tool Adjusting scan speed e Use the Scan Speed slider in the Acquisition Mode tool Fig 18 to adjust the scan speed A higher speed with averaging results in the best signal to noise ratio Scan speed 8 usually produces good results Use speed 6 or 7 for superior images Choosing the dynamic range e Select the dynamic range 8 or 12 Bit per pixel in the Bit Depth pull down in the Acquisition Mode tool Fig 18 8 Bit will give 256 gray levels 12 Bit will give 4096 gray levels Publication quality images should be acquired using 12 Bit data depth 12 Bit is also recommended when doing quantitative measurem
5. Application Window the Center Screen Area Each displayed image can be displayed and or analyzed with many view options available through view tabs which can be found on the left side of the image According to the chosen view tab the required view controls appear in View Control Tabs below each image File management and data handling tools are found in the Right Tool Area see Fig 4 and Fig 5 Color and brightness of the interface have been carefully adjusted to the typical light conditions of the imaging laboratory guaranteeing optimal display contrast and minimal stray light for high sensitivity detection experiments The ZEN software is optimized for a 30 TFT monitor but can also be used with dual 20 TFT setups E ir iimm Show all mode active are er ron Ona i jajem bow Tiger bh Tripper Cel aa meg m 10 eee fiigail Trig F Show all mode inactive af Tree Cal 2 hin Fig 6 Basic and Pro Mode A focus in the development of ZEN 2009 was to fulfill the needs of both basic users and microscopy specialists Both types of users will appreciate the set of intuitive tools designed to make the use of a confocal microscope from Carl Zeiss easy and fast The Show all concept ensures that tool panels are never more complex than needed With Show all de activated the most commonly used tools are displayed For each tool the user can activate Show all mode to display and use additional functionalit
6. ack LSM 12 10 2009 Settings for track configuration in Channel Mode e Select Channel Mode if necessary Fig 15 m Imaging Setup V Show sit A The Light Path tool displays the selected track mE iainih Yo simultaneous configuration which is used for the scan procedure Switch track every Frame e You can change the settings of this panel using the V Tadi following function elements Fig 15 Imaging Setup tool for a single track LSM Activation deactivation of the excitation wavelengths check box and setting of excitation intensities slider If necessary open the Laser Control tool see above Selection of the main dichroic beam splitter HFT or 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 Activation deactivation via check box of the selected channel Ch 1 4 monitor diode ChM META detectors ChS1 8 transmission ChD for the scanning procedure and assigning a color to the channel e Select the appropriate filters and activate the Imaging Setup Som chan nels Firde Chenn e T amane e Click the Laser icon to select the laser lines and Switch back every Frema set the attenuation values transmission in in AFapa TdTo the displayed window AF488 Td Peh e For the configuration of the beam path please refer to the application specific configurations
7. available in the List of Tracks panel in the Imaging Setup Tool Fig 15 Fig 16 and Fig 17 Switch track every Line Tracks are switched during scanning line by line The following settings can be changed between tracks Laser line laser intensity and channels Frame Tracks are switched during scanning frame by frame The following settings can be changed between tracks Laser line and intensity all filters and beam splitters the channels incl settings tor gain and offset and the pinhole position and diameter Frame Fast The scanning procedure can be made faster Only the laser line intensity and the Amplifier Offset are switched but no other hardware components The tracks are all matched to the current track with regard to emission filter dichroic beam splitter setting of Detector Gain pinhole position and diameter When the Line button is selected the same rules apply as for Frame Fast An additional track is added to the configuration list in the Imaging Setup Tool The maximum of four tracks can be used One track each with basic Add Track button configuration is added i e Ch 1 channel is activated all laser lines are switched off emission filters and dichroic beam splitters are set in accordance with the last configuration used ma Remove burton The track marked in the List of Tracks panel is deleted A A click on this arrow button will move the selected track highlighted in light grey one position upwards in the
8. button changes to a Cancel button Click Cancel to interrupt stop the Startup of the system r Startup the ZEN Main Application window Fig 4 and Fig 5 opens To benefit from all of Zen s features run the window in its full screen mode 10 2009 3 4 Application Bar E Whenu Bar C fam Toolbar 6 Lett Tool Ares E Center Screen Area hosts up ta 3 I Containers F Right Tool Area G Status Area Fig 4 ZEN Main Application window after Startup with empty image container 1 F 3 4 4 10 5 1 Tabs te switch between the tool types 6 View tabs 2 Start buttons 7 image tabs 3 Tool group 8 Displayed image 4 Tool 9 File handling area 5 Status bar 10 View contrals Fig 5 ZEN Main Application window after Startup with several images loaded 10 2009 Introduction to ZEN Efficient Navigation The ZEN 2009 interface is clearly structured and follows the typical workflow of the experiments performed with confocal microscopy systems On the Left Tool Area Fig 4 D the user finds the tools for sample observation image acquisition image processing and system maintenance easily accessible via four Main Tabs Fig 5 1 All functions needed to control the microscope can be found on the Ocular Tab to acquire images use the Acquisition Tools Fig 5 3 and 4 Arranged from top to bottom they follow the logic of the experimental workflow The area for viewing and interacting with images is centered in the middle of the Main
9. current config can be deleted by pressing the delete button These configurations can be assigned to buttons that are easier to press L Depending on the microscope configu ration settings must be done manually if necessary 10 2009 Transmitted light control Reflector Reflected light shutter Reflected light Source X Cite 120 or HBO 100 Fig 12 Microscope Control window with Transmitted Light pop up menu Confirman Assign FF GFP Fig 13 Configuration panel 11 Configuring the beam path and lasers e Click on the Acquisition button Acquisition Setting up a configuration Simultaneous scanning of single double and triple labeling Advantage faster image acquisition Disadvantage Eventual cross talk between channels Sequential scanning of double and triple labeling line by line or frame by frame Advantage Only one detector and one laser are switched on at any one time This reduces cross talk Disadvantage slower image acquisition e Open the Imaging Setup and the Light Path tool in the Setup Manager Tool group to access the hardware control window to set up the beam path The open Light Path is shown in Fig 14 Birri Tita Sach aor hey Tis aa Pre otis m ji fers ies Seton Minap E D 7 mepa ii T Light Pai Dfii Aura ec m Aue ine i Chaman Hak Spee Apatan inion On Exper A Sue Sa r gt Fig 14 Light Path tool for a single tr
10. depending on the used dyes and markers and the existing instrument configuration AFABE TaTo e n the Imaging Setup tool the Detection Bands amp Laser Lines are displayed in a spectral panel Fig 16 Fig 16 to visualize the activated laser lines for excitation vertical lines and activated detection channels colored horizontal bars Detection Bands amp Laser Lines display 10 2009 13 ae zal de e For storing a new configuration click m and enter a desired name in the first line of the list box Fig 17 then click Ok to store the con figuration Save cumenti acquistion configuration as Configuration e For loading an existing configuration click Ok Cancel then select it from the list box e For deleting an existing configuration click pes Fig 17 Track Configurations window then select it from the list box and confirm the deletion with Ok Settings for multiple track configurations in Channel Mode Multiple track set ups for sequential scanning can be defined as one configuration Channel Mode Configuration to be stored under any name reloaded or deleted The maximum of four tracks with up to eight channels can be defined simultaneously and then scanned one after the other Each track is a separate unit and can be configured independently from the other tracks with regard to channels Acousto Optical Tunable Filters AOTF emission filters and dichroic beam splitters The following functions are
11. detector gain and offset Use one of the Auto Exposure Live Continuous or Snap buttons to start the scanning procedure to acquire an image Scanned images are shown in separate windows Click on the Stop button to stop the current scan procedure if necessary Select Auto Exposure for Select Live for continuous fast scanning useful for finding and changing the focus Fig 20 Image Display Select Continuous for continuous scanning with the selected scan speed Select Snap for recording a single image Select Stop for stopping the current scan procedure Choosing Range Indicator gt Clicking on the right hand side of the In the View Dimensions View Option 1 om Control Block click inside the color field in the 00 vung L button under the channel button Fig 21 button leads to a list of colors Fig 21 View Dimensions Control Block 10 2009 17 Fig 22 Image Display Channels racks Wf Tracki Select all Unselect all Tracki wal vw 456 505 S61 633 n 6 5 p Continuous Wawe Attenuation OFF 70 z 1 0 i 32 0 i TAU max aif 0 1 0 435 0 1 0 610 0 10 Fig 23 Channels tool 18 The scanned image appears in a false color presentation Fig 22 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 intensit
12. e 1 Poo Bi mer cals ce E imreChi E fete l L Cee bree GF Him ieai z Mia Hi m Pogum Terei on ELF INIP reel ete at Joni Borah Caife L 4 foo Bo Mizar a al BA i AAE Daen bar Fea a a jmi A E Cehi Ur BEET PLCS E Apo flea E baira Ari 88 kora a daiji 10 ci E Aike Smh LEK G r PEPES DU h i File Browser LEhg reread 2 Sh eri res ba hij OA ae oO AE orice bree a Chere be bii G12 ae fea ee Bl Cs gi l Chera Ahi O TIAE rEg i i rmh g ed mA 3 Chenraii 2 61 PS ho hia eR re ee eet 3 Chrertren te kii amp a k fa DST ie gt Cire ree hii TANS i SE ae IChenmeis Oiri LEHE LST NE PT E a irs ICHE is Bate UET AE CENTE ee E ee 1 Chenreis F bti oe a5 ine pirre ee ke ery mimi u U age TE kA RE pire Pere gt Chern is amp bel D14 MB CETNA Gim MAOT 3 Chemi ti ETIN E Epes i hub cl ee Cherin 3 br LTI HA HEET iiher AETHON 3 Cheia a i IFN ee hiesari di 3 Chen raii bii G34 11 HE beninn 3 Ch eriree i 8 5 Pet TIIME Cai ph ri Iche ba bii A CETHE akh sirat Chera Ahi 0 TIAE LTA barano 3 Chenreie 2 H ApS Eeg om Pe ed ered ot Ch gq 3 Chenrais kii nit ae Det we Dart gt Cire rete 2 bH 10 2009 Turning on the lasers e To manually switch lasers on or off open the Laser tool e All available lasers can be operated within t
13. ents or when imaging low fluorescence intensities 10 2009 15 Setting scan averaging Averaging improves the image by increasing the signal to noise ratio Averaging scans can be carried out line by line or frame by frame Frame averaging helps to reduce photo bleaching but does not give quite as smooth of an image e For averaging select the Line or Frame mode in the Acquisition Mode tool e Select the number of lines or frames to average Adjusting pinhole size e Select the Channels tool in the Left Tool Area e Set the Pinhole size to 1 AU Airy unit for best compromise between depth discrimination and detection efficiency Pinhole adjustment changes the Optical Slice thickness When collecting multi channel images adjust the pinholes so that each channel has the same Optical Slice thickness This is important for colocalization studies Channels Tracks A Tracki Select all Unselect all w vw 405 488 505 561 633 405 nm iets tied i Continuous Wave Attenuation OFF Conia ah i r ms r ms TAU max Tia Ear Fite nmm hey Tiin Bian ai m iad LUPO Vins etp Mira Latii F mepa iip F Lihi Pai Dra Aput m Arjus ire harren Hii Baran rE Apan hemin On Ean E An Save Fig 19 Channels tool 16 10 2009 Image acquisition Once you have set up your parameter as defined in the above section you can acquire a frame image of your specimen a automatic pre adjustment of
14. face For a more detailed description of the functionality for the ZEN 2009 software please refer to the User Manual that is provided with your system 10 2009 a To create a new image document in an empty image container click the Snap or the Auto Exposure button For an empty image document press the New button The new document is immediately presented in the Open Images Area Remember an unsaved 2D image in the active image tab will be over written by a new scan Multi dimensional scans or saved images will never be over written and a new scan will then automatically create a new image document Acquired data is not automatically saved to disc Make sure you save your data appropriately and back it up regularly The ZEN software will ask you if you want to save your unsaved images when you try to close the application with unsaved images still open L There is no image database any more like in the earlier Zeiss LSM software versions z ai io Sr eee BE Al La a ee es a pe ips 10 2009 Advanced data browsing is available through the ZEN File Browser Ctrl F or from the File menu The File Browser can be used like the WINDOWS program file browser Images can be opened by a double click and image acquisition parameters are displayed with the thumbnails Fig 9 For more information on data browsing please refer to the detailed operating manual Pt eal J Fig 9 uo Hy Dees Piy Feirm E a LE 1 Amm
15. his tool Fig 10 Fig 10 10 2009 Laser Laser Laser Lines nm Argon 458 477 488 514 HeNe5gad HeNe543 HeNe633 Laser Properties Maximum Power 30 0 mW Wavelength 458 477 466 514 nm Status Waning up Tube Current 39 A Output Laser Control tool Power On On OF On Setting up the microscope Changing between direct observation camera detection and laser scanning mode The Ocular Camera and LSM Acquisition buttons switch between the use of the LSM and the microscope the beam path and indicate which beam path is currently in use for the microscope aa EP BDL ie Cogiraion aan ta i s as Ocular EC Plan Neofhuar 1t0D_3 Aperture 0 16 HF Fig 11 Microscope Control window e g Axio Imager Z1 e Click on the Ocular button to change open the controls for the microscope beam path and for direct observation via the eyepieces of the binocular tube lasers are blocked e To set the hardware in position for using the microscope click Online if not yet active e To close the light shutters on the microscope click Offline e Click on the LSM Acquisition button to move back to the LSM system Setting up the microscope and storing settings Click on the Ocular tab for direct observation press the Online button for your actions to take effect immediately Then open the Ocular tool to configure the components of your microscope like filters shutters or objec
16. overlays choose Export from the File Menu In the Export window you can select from a number of options and proceed to the WINDOWS Save As window to save the exported data to disk 10 2009 Switching off the system Click on the File button in the Main Menu bar and then click on the Exit button to leave the ZEN 2009 software If any lasers are still running you should shut them off now in the pop up window indicating the lasers still in use Shut down the computer Wait until the fan of the Argon laser has switched off On the REMOTE CONTROL turn off the Components switch and the System PC switch Fig 1 Switch off the X Cite 120 lamp or the HBO 100 mercury burner Switch off the UV Ar laser of by the toggle switch on the power supply Fig 2 10 2009 21
17. r supply It will be ready for operation after a few seconds Fig 2 Power supply of UV Ar laser 5 10 2009 Starting the ZEN software ZEN Ed FEN 2009 e Double click the ZEN 2009 icon on the WINDOWS desktop to start the Carl Zeiss LSM software The ZEN Main Application window and the LSM 510 Startup window appear on the screen Fig 3 Laga IEN SEN Lami Eee j Arie Lo I E REN mA Ay Ei kpn rh AEF Lam See Gece LL a ZEN 2009 Main Application window ire Ar c LSM 510 Startup window Fig 3 ZEN Main Application Window at Startup a and the LSM 510 Startup Window b and c In the small startup window choose either to start the system Start System hardware for acquiring new images or in Image Processing mode to edit already existing images Toggle the little o symbol to view the Boot Status display and get the additional Offline Demo button option Afte Choosing Start System initializes the whole microscope system and activates the entire software package for new image acquisition and analysis The Image Processing mode ignores all hardware and activates only data handling and image processing functionality for already acquired images The Offline Demo mode reads the current hardware database but does not activate the system hardware for use Instead it simulates the system hardware for training purposes Upon clicking the Start System button the Image Processing
18. tives Fig 11 Selecting an objective Open the graphical pop up menu by clicking on the Objective symbol and select the objective lens for your experiment Fig 11 The chosen objective lens will automatically move into the beam path Focusing the microscope for transmitted light Open the graphical pop up menu by clicking on the Transmitted Light icon Fig 12 Click on the On button Set the intensity of the Halogen lamp using the slider Clicking outside the pop up control closes it Place specimen on microscope stage The cover slip must be facing the objective lens Remember the immersion medium if the objective chosen requires it 10 2009 e Use the focusing drive of the microscope to focus the 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 icon to open the X Cite 120 controls and turn it on e Click on the Reflected Light shutter to open the shutter of the X Cite 120 lamp HBO100 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 as configurations Fig 13 by typing a config name in the pull down selector and pressing the save button Fast restoration of a saved config is achieved by selecting the config from the pull down list and pressing the load button The
19. y Fig 6 10 2009 5 Workspace zoom E Light Path Channel Mode Lambda Mede Undock tool Fig 7 ZEN Window Layout configuration More features of ZEN 2009 include The user can add more columns for tools to the Left Tool Area or detach individual tools to position them anywhere on the monitor To add a column drag a tool group by the title bar e g Online Acquisition to the right and a new tool column automatically opens Alternatively use the context menu move toolgroup to next column To detach a tool click on the little icon on the very right end of the blue tool header bar Fig 7 Another unique feature in Imaging Software is the scalable ZEN interface This Workspace Zoom allows adjustment of the ZEN 2009 window size and fonts to the situational needs or your personal preferences Fig 7 Setting up conventional confocal software for a specific experiment can take a long time and Is often tedious to repeat With ZEN these adjustments have to be done only once and may be restored with just two clicks of the mouse For each type of experiment one can now set up and save the suitable Workspace Layout These configurations can also be shared between users For most controls buttons and sliders a tool tip is available When the mouse pointer is kept over the button a small pop up window will display which function is covered by this tool outton These are just some of the most important features of the ZEN inter
20. y e Set the Pinhole to 1 Airy Unit Fig 23 e Set the Gain Master high e When the image is saturated reduce AOTF transmission in the Laser control section of the Channels Tool Fig 23 using the slider to reduce the intensity of the laser light to the Specimen Adjusting gain and offset e Increase the Digital Offset until all blue pixels disappear and then make it slightly positive Fig 23 e Reduce the Gain Master until the red pixels only just disappear 10 2009 Scanning a Z Stack A continuous XY scan of the set focus position will be performed Select Z Stack MESEL in the main tools area Open the Z Stack tool in the Left Tool Area Select Mode First Last on the top of the Z Stack tool Click on the Wik button in the Action a Interea Button area tl Chl ETH ko CHN SELH ami ep Use the focus drive of the microscope to focus on the upper position of the specimen area where the Z Stack is to start Ho Comec Click on the Set First button to set the upper Pg 2A a i position of the Z Stack Then focus on the lower specimen area where the recording of the Z Stack is to end Click on the Set Last button to set this lower position Click on the button to set number of slices to match the optimal Z interval for the given stack size objective lens and the pinhole diameter P Start Experiment Click on the Start Experiment button to start the recording of the Z Stack
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