information on this system.
Contents
1. Ciianiraia Oiri LEHE LST NE mer ben LE ROME PE J Chtaniraiia 8 Gi D41 NE LET NE EB ee TE Emi I Nr AITWE a5 pw Petre Lima eed rry 2 Chan res is dn TE LN Ken ipn liga Charmes E br 014 UB CETNA dai HETT 3 haniais dbi D13MB Freie prxcirrra d ub cd ua Chen 2 br LTI HA 79 MB iiher FETT LI d e 3 Chwinala 8 4 15 kH D4JI WE hiesari uli 3 Chen raii 3 bij CES MA 11 48 vertan Em EN I AE HH EEA HE Luiphri Iche la 4 bii Av kn CETHE akh sirat Chera A bil Ii MI ME 1 1 af arene 5 Liianraia E Erl EE EH Eeg om ILI nr be en rag ka 3 Chaniraia kii nit ME DET S Dart 3 Cire nata 3 bil 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 this tool Fig 10 Fig 10 10 2009 Laser Laser Laser Lines nm Argon 458 477 488 514 HeNe594 HeNeb543 HeNe633 Laser Properties Maximum Power 30 0 mW Wavelength 458 477 488 514 nm Status Warming up Tube Current 3 9 A Output 56 Laser Control tool Power On On Off On Setting up the microscope Changing between direct observation camera detection and laser scanning mode The Ocular Camera and LSM Acguisition 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 FREFES2. 30 20 10 Wavelength nm B ET426 2 0x E T455LP B ET480 40m httos www micro shon zeiss com n us amp f f httn www chroma com nroducts catalos 49000 Series Filter Sets 49002 ET GFP FITC Cy2 Wavelength nm B ET470 40x B T455lp lt r B ET525 50m 49003 ET EYFP 100 90 50 70 60 a 40 30 20 10 0 Wavelength mm B ET500 20x B T515LP E ET535 3 0m httos www micro shon zeiss com n us amp f f htto www chroma com nroducts catalog 49000 Series Filter Sets Filter Set 45 000000 1114 462 r ar kal D T E m fen jon E m m L m D ar I I rm im Lu Fog anu Havelength Inn Filter Set 45 B Excitation BP 560 40 L V Beam Splitter FT 585 B Emission BP 630 75 httos www micro shoo zeiss com 0 us amp f f htto www chroma com products catalos 49000 Series Microscope Quick Guide Zeiss LSM 510 Axiolmager M1 Microscope Guide ZEN Room 4502 Start Up Procedure 1 Remove dust cover from microscope 2 Toggle X Cite Lamp switch ON 3 On Remote Control Pad toggle the System PC switch and the Components switch ON 4 Always leave Power Supply 231 switch ON position 5 Turn Computer ON 6 User name New User no password 7 Open program ZEN 2009 8 Press Start System button to initialize hardware Lasers 1 Click Acquisition tab Under Setup Manager heading click on Lasers to open the lasers too
3. dual 20 TFT setups Bl tre Show all mode active Lt kr reve Caras faren ban Tiger bh Hr CERT apum ra 7 nn fingrer Tri show all mode inactive af Tree Ca M hire 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 functionality Fig 6 10 2009 gt Workspace zoom E Light Path Channel Mode Lambda Made 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
4. ETE ETE n kbps ud ae planet Ful mectitri rrage Sy El 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 and 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 th
5. 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 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
6. Start System hardware for acquiring new images or in Image Processing mode to edit already existing images Toggle the little LJ 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 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 A Application Bar B Menu Bar C Main Toolbar D Left Tool rea E Center Screen Area hosts up ta 3 1 Containers F Right Tool Area G Status Area Fig 4 ZEN Main Application window after Startup with empty image container 1 2 3 E a 10 5 1 Tabs to switch between the tool types amp View tabs 2 Start buttons 7 Image tabs 3 Tool group B Displayed Image 4 Tool 5 File handling area 5 Status bar 10 View controls Fig 5 ZEN
7. and press Save Reuse Button 1 2 To capture a new image using the exact acquisition parameters pinhole diameter Gain Master Digital Offset excitation beam path scan mode frame size speed data depth scan direction average zoom of an existing image click Reuse button on the bottom panel of an opened image The acquisition parameters of a displayed image can be viewed by clicking on Info button on the left of the image Shut Down Procedure 1 2 3 If used oil lens thoroughly remove oil using dry Lens Paper Put 10x objective in place Put Argon laser on standby but leave other lasers on Continue if last user 4 O ON Du From the Lasers Tool turn lasers OFF Close the ZEN program Shut down computer Wait for computer to turn off Always leave Power Supply 231 Box switch ON On Remote Control Pad toggle the System PC switch and the Components switch OFF Toggle X Cite Lamp switch OFF 10 Cover microscope with dust cover EET 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 fM i ai a io iai aid a a i aaa a i ias oe e 1 hode 1 St rting the SYSTE EEE PE en 2 Introduction to ZEN Efficient Navigation annnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnner 5 Setting up the MICFOSCODE Las same peike 10 Configuring the beam path and lasers 12 Seng AN I
8. current scan procedure Choosing Range Indicator ES Clicking on the right hand side of the In the View Dimensions View Option i is E Control Block click inside the color field in the LM vung Ld 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 I racks v Tracki Select all Unselect all Track1 vi vo v 488 505 561 633 i 6 5 s Continuous Wave Attenuation OFF 70 1 0 3 320 i TAU max aif 0 1 0 435 0 1 0 610 0 1 0 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 intensity 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 wi
9. E ETE BDL ie once iE gn Zur amp 1 mE Y Ocular EC Plan Neofhuar 10 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 objectives 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 sip must be facing the objective lens Remember
10. NE aaa aisiais 15 Storing and exporting image data rennnnnrnnnnnnnrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnennnnnnennnnnnnennnnnnnennnnnnnunnn 20 Switching off the SEN a ee i 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 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 syste
11. 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 button These are just some of the most important features of the ZEN interface 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 n 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 ap
12. Zeiss LSM 510 Axiolmager M1 Confocal Microscope 2 A er lt Objectives Zeiss LSM 510 Axio Imager M1 Microscope Objectives 12 1 2 gt EC Plan Neofluar 10x Plan Apochromat 20x EC Plan Neofluar 40x 0 3 0 8 1 3 Oil 10x 20x ICS ICS ICS c Room 4502 Plan Apochromat 63x Achroplan 40x W Plan Apochromat 63x 1 4 Oil 0 8 W 1 0 VIS IR 63x 40x 63x DIC III DIC III Catalog 420780 9900 440090 9901 ICS Infinity Color Corrrected System Filter Sets Zeiss LSM510 Axiolmager M1 Microscope Filter Sets Room 4502 1 Transmitted Light 2 Zeiss Filter Set 02 DAPI 3 Chroma Filter Set 49001 CFP Excitation BP 436 20 Emission BP 480 40 Similar to Zeiss Filter Set 47 4 Chroma Filter Set 49002 GFP Excitation BP 470 40 Emission BP 525 50 Similar to Zeiss Filter Set 38 5 Chroma Filter Set 49003 YFP Excitation BP 500 20 Emission BP 535 30 Similar to Zeiss Filter Set 46 6 Zeiss Filter Set 45 Cy3 5 mcherry Excitation BP 560 40 Emission BP 630 75 Filter Sets Filter Set 02 455002 9901 000 Pr DE D z E m al E r m m a nm E Ti z E D Is T rl m Kjem Lu 700 enu Havelength Inn Filter Set 02 Excitation G 365 LI L V Beam Splitter FT 395 LI V Emission LP 420 49001 ET CFP i 90 80 sof 40
13. aser 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 Jaer chan nels Foch Channel hode T ES EET IEEE e dE e Click the Laser icon to select the laser lines and Switch track every Frame T set the attenuation values transmission in 96 in arsss TdTo the displayed window AF488 Td Au e 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 AFABB 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 EMEN sir fe 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 curren
14. e 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 Zeiss LSM 510 Axiolmager M1 Rm 4502 Start Up Procedure 1 Remove dust cover from microscope 2 Toggle X Cite Lamp switch ON 3 On Remote Control Pad toggle the System PC switch and the Components switch ON 4 Check if the Power Supply 231 box switch is ON 5 Turn Computer ON 6 User name New User no password 7 Open program ZEN 2009 8 Press Start System button to initialize hardware Shut Down Procedure 1 If used oil lens thoroughly remove oil using dry Lens Paper 2 Put 10x objective in place 3 Put Argon laser on standby but leave other lasers on Continue if last user 4 From the Lasers Tool turn lasers OFF 5 Close the ZEN program 6 Shut down computer Wait for computer to turn off 7 Leave the Power Supply 231 Box switch ON 8 On Remote Control Pad toggle the System PC switch and the Components switch OFF 9 Toggle X Cite Lamp switch OFF 10 Cover microscope with dust cover CB IACore F Facility Attention Users X Cite Fluorescence Lamp The X cite lamp must be on for at least 30 minutes before being turned off AND must not be turned on again within 1 hour of being turned off 1 When starting before you turn on the X Cite lamp make sure it has been OFF fo
15. er 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 Remove Button 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 list box v 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 A The number of pixels influences the image resolution Acquis
16. ew image For the Range Indicator click on the Channel Color found under your image window MN o Red saturated Blue zero pixels Adjust your Gain Master such that a bit of red overexposure is seen and then adjust the Digital Offset so that minimal blue zero pixels is obtained Click Stop button Once all channels have been adjusted re activate all of your channels tracks Click Snap to capture your final image Microscope Quick Guide To Capture a Z stack 1 ple ee Na 10 Click on the Check the Z stack option in the main tools area Open the Z stack Tool found under the Multidimensional Acquisition heading Make sure that First Last mode is selected Click on Live button to get a preview image with only one active track Turn Fine Focus knob CW to an optical plane for starting Z stack capturing and click Set First button Turn Fine Focus knob CCW to an optical plane to be the last optical section of the Z stack and click Set Last button Click Stop button Re activate all of your channels Optima Click on the button to set number of slices to match the optimal Z interval for a given stack size objective lens and pinhole diameter P Start Experiment button to start acquiring your Z stack To Save an Image 1 2 To save an image click File Save as or click the button in the Main Toolbar or the EJ button in the File Handling Area Enter a file name select the LSM 5 Ism format
17. ition Mode Objective EC Plan Neofluar 10x 0 3 Scan Mode Frame r Frame Size X 128 Y 128 Optimal speed Pixel Dwell 640 psec Scan Time 491 52 msec Averaging Number Bit Depth S 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 guality images should be acguired using 12 Bit data depth 12 Bit is also recommended when doing guantitative measurements 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 comp
18. l 2 Turnon the required laser s for your study Toturnon the Argon 2 laser select Standby first Click on laser properties at bottom of the Laser Tool After about 1 minute of warming up status will change from warming up to ready Change Output to 50 Select On The HeNe543 and HeNe633 lasers can be turned on directly by selecting On Configuring Microscope Mode 1 You can load pre configured tracks in the Imaging Setup Tool Click on the open icon to select an optical track To add another track click the button to add a new empty track and then use the open icon to select a pre configured track to replace the empty track H Imaging Setup V Showal Mode Channel Mode v simultaneous Switch track every Frame v V AF488 TdTo A488 tdTo ERTER LU BEL AF 488 TdTo Open the Light Path Tool You can toggle between the different tracks found in the Imaging Setup Tool to view the respective optical configuration in the Light Path Tool For each track click on Lasers button found in the Light Path Tool and set the desired Transmission for each Laser Recommended Argon 2 5 10 HeNe633 5 10 HeNe543 50 80 Microscope Quick Guide Configuring Scan Parameters 1 2 3 Open the Acquisition Mode Tool located under the Online Acquisition heading Select the appropriate objective from the drop down list Select the appropriate Frame Size ie 1024x1024 o OR click Optimal button to get a f
19. lab folder your folder Files found on anywhere else will be deleted daily You are responsible for backing up your own data at all times Do not rely on this computer as storage for your data Once you have backed up your files elsewhere please delete your files from this computer How to connect to the CBIA CORE network C MEDTECH SERVICE DES TECHNOLOGIES TECHNOLOGY SERVICES CBIA CORE To connect to the shared folder CBIA CORE click on this icon n the desktop or click on start all programs Medtech and medmapdrives You will be prompted with your UOttawa computer username and password DO NOT put a check mark in Sauvegarde Save MedMapDrives DFS Bienvenue Welcome Cet outil vous permettra de vous brancher au r seau de la Facult de m decine afin d acc der aux dossiers partag s ainsi qu votre espace personnel This tool will allow you to connectto your shared and personal drives on the Faculty of Medicine network Authentification Authentication Veuillez utiliser votre nom d usager et mot de passe pour votre compte la Facult de m decine pour S acc der vos disques Please use your Faculty of Medicine username and password to access the shared drives MEDTECH SHIVICE DES TECHNOLOGIES I ECHNOLOGY SERVICES Nom d usager Username Mot de passe I Password Sauvegarder Save Then go to My Computer R drive Research CBIA Core your Lab Folder When you are do
20. ll 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 BE button in the Action Button area tl ERI ETH ko Sei Se Hoen and Sep Use the focus drive of the microscope to focus on the upper position of the specimen area where the Z Stack is to start Jure Click on the Set First button to set the upper SS ae 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 gt Start Experiment Click on the Start Experiment button to start the recording of the Z Stack ES When a multi dimensional acquisition tool is not selected the respective tool and its set parameters are not included in the multidimensional image acguisition 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 ET mass kia Zrabash et den Fao ES Save ai IE LEM Sf trai Cancel x Fig 26 Save as window Tapp Irraza Pru Comms of Fraga mrin rija pans Coes ox FLAGS
21. m 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 power supply It will be ready for operation after a few seconds Fig 2 Power supply of UV Ar laser gt 10 2009 Starting the ZEN software ZEN Ed ZEN 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 IN IE REF Lami Eee ie Arie et E REN NS ae Tate Langen TTH ME I i a ILES Gece LL a ZEN 2009 Main Application window ire Ans 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
22. ne you HAVE to disconnect the drives Go to my computer right click on each of the three drives Pool Research Web and select disconnect u Ottawa Facult de m decine Faculty of Medicine RGN 2129 e Telephone 613 562 5648 e medtech uottawa ca
23. propriately 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 ES There is no image database any more like in the earlier Zeiss LSM software versions nu m ua m E A DEI ue quacum TA E LII xa T pe 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 er Fig 9 Ka v Dees By Feirm Denk farser LE Poo Bi mer cals D od B pe E Mac Cool DELE LAE GF Duane ll fiken Mas KSS Pogum Basi vds Vere ELF INIP brani rx Joni kiti Tech Cort LG Frem bai Mizar ur al BA AM Later Obf Fea an tire a An dires Data mr BEET HLCEE re flea E baira Kerken 48 core a vdi 40 coke EB Eee Aike rl LC Ds Dr Li LAA File Browser k A amp Ehgirars t EE I 3 bil E14 ae HEIME us baren ar Chere la 3 bil E 123 ae DEN NE L Cia 11 Hair iun ta A Li i Tl ss hia re g muni oe ba 5 Chanrais B brl PS RH ERE I Li uu He eet E 3 Chanria iz 3 bii E LA k 5 DST ie gt Cire ree 3 bil LA ME n EH ME
24. r at least 1 hour from previous use 2 When finishing before you turn off the X Cite lamp make sure it has been ON for at least 30 minutes Booking Time You are allowed to sign up for a maximum of 3 hours per day If no one signs up after you you can continue to use the station until someone else needs it You are responsible for showing up for your time booked A 10 00 fee applies if you did not show up for booked time Log Book All users must log the time spent on the station as well as the time the fluorescence lamp was turned on off in the log book NO FOOD OR DRINKS PERMITTED Attention Users Immersion OIL Rules e Always keep immersion oil bottle CLOSED This prevents water evaporation and maintains the integrity of the oil Keep oil bottle CLEAN Oil should never be found along the sides of the bottle or around the neck Apply only a SMALL AMOUNT of oil to your coverslip Oil droplet Cove rslip Always load your sample on the microscope stage with the STAGE LOWERED to the lowest focus position Always REMOVE OIL using dry Lens Paper from the objective when changing slides Re apply new oil to the new slide e f you are imaging a sample using an oil objective do not toggle between objectives Image with that objective only e When you are done your session Wipe off oil objective using Lens Paper e Do not get oil on any air objectives Your Data Save all of your work in F drive Data your
25. rame size appropriate for Nyquist Sampling Select the appropriate Scan Speed ie 5 9 For Averaging to increase Signal Noise ratio Number 2 or 4 Bit Depth Set as desired Mode Line Direction gt gt Method Mean Set Sample on Stage and Focus A 1 2 3 4 To be able to view specimen down ocular need to first click Occular Tab then Online Lightly wipe your slide on both sides with a kimwipe and a small amount of 70 ethanol to clean If using an oil lens add a very small drop of immersion oil Set slide on stage bring specimen into focus and select desired area of interest Click on Acquisition tab diusting Channels and Capturing an Image E ad Eum Fi Select Auto Exposure for 5 Select Snap for recording a Auto Exposure automatic pre adjustment of Snap single image detector gain and offset Select Live for continuous fast a Select Stop for stopping the scanning useful for finding and current scan procedure changing the focus Select Continuous for continuous scanning with the selected scan speed Continuous Open the Channels Tool found under the Online Acguisition heading For each channel set the pinhole size to correspond to 1 Airy Unit as a starting point Adjust the pinhole sizes to get the same resulting optical section thickness across all channels Perform the following for each individual channel temporarily deactivate other channels Click Live button to get a previ
26. romise 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 V Tracki Select all Unselect all vi v xv 405 488 505 561 633 405 nm em ka ini i Continuous Wave Attenuation OFF Canin 42H i al ms r ms r 1AU max Te Ear Fite nmm h rt Tila ar MAE Ti ai Ti Stop Manager Latii imagen ZaiLio F Leah Patti Dra Aa rer roues re harme zu Br imme TETE Arpa II hemin inn BU Aum Save Fig 19 Channels tool 16 10 2009 Image acguisition Once you have set up your parameter as defined in the above section you can acguire a frame image of your specimen Pe automatic pre adjustment of 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
27. t acquisiion 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 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 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 for gain and offset and the pinhole position and diameter Frame Fast The scanning procedure can be made faster Only the las
28. 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 current config can be deleted by pressing the delete button These configurations can be assigned to buttons that are easier to press ES 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 Gondegpunraben Assign Fr CFP Fig 13 Configuration panel 11 Configuring the beam path and lasers e Click on the Acquisition button Acquisi
29. tion 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 hrs Tic Peri rina h rt Tii Sata NAT fers is tup Manager E Fi mepa SD 5 Light Patti Drin Aura En Arguesshn re NEM Chaman Hark Spee Aas pein inion On Exper 3 i ru us r Fig 14 Light Path tool for a single track 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 PE 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 Track 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 L
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