LSM 780 Quick Guide
Contents
1. Multidimensional Acquisition Z Stack Tile Scan i Information On Experiment IB Auto Save Fig 10 11 2013 V_03 amp Laser P Start Experiment Mai Tai Argon DPSS 561 10 HeNe5o4 HeNe633 Diode 405 30 Laser Properties Maximum Power Wavelength Status Laser Control tool 690 1024 458 488 514 561 594 633 F 5 0 mw 633 nm connected Setting up the microscope Changing between direct observation and laser scanning mode The Locate and Acquisition buttons switch between the use of the LSM and the microscope Click on the Locate button to open the controls for the microscope beam path and for direct observation via the eyepieces of the binocular tube lasers are blocked Ocular Incubator A e Click on the Acquisition button to move back to the rre LSM system DAPI Smart Setup Show all Tools New AF O E oa Find Focus Set Exposure Continuous Snap 10 11 2013 V_03 Setting up the microscope and storing settings Click on the Locate tab for direct observation press the Oculars 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 e Open the graphical pop up menu by clicking on the Objective symbol and select the objective lens for your experiment Fig 11 e The chosen objective lens will automatically m2. Current Position Sample Carrier Position List Calibrate E Single positon Fig 36 Positions tool 29 Start and monitor the experiment with the Action buttons Fal Count rate xyz SCan Press the New button to open a new FCS diagram into an image container If a measurement Is triggered all data are displayed in that window if highlighted Press the Count rate button to open the real time display window for the detector Count rate in all active channels This allows you to optimize your experiment by changing the laser power and the pinhole size while monitoring the count rate Press the xyz Scan button to display the current coordinates You can define boundaries where a scan is performed with simultaneous acquisition of the count rate This allows you for example to identity labeled molecules accumulated in the membrane Press the Snap button to trigger one measurement at the highlighted or first defined position Press the Start ConfoCor Experiment button to trigger a measurement All defined positions will be approached consecutively Press the Stop button to end a measurement All data accumulated so far will be available and can be stored After completion of the measurement the data will be displayed in the FCS Correlation diagram within an Image Container see Fig 37 11 2013 V_O3 0 00010 0 0003 0 0010 0 003 0 010 0 03 ulse distance ms 1e 006 0 0001 Rh6G cys Rh6G Repetition Channe C
3. Fig 14 Smart setup tool Once finished with the input Smart Setup suggests four alternative considerations see below One for Fastest imaging one for the Best signal Smartest Line between both speed and best signal and the optimal setup for later Linear unmixing of the dyes The graphs display relative values for the expected emission signals and cross talk The resulting imaging scheme single or multitrack is shown below the graphs 11 2013 V_03 13 Show all Smart Setup Dye Cy5 Alexa Fluor 488 TRITC DAPI Current Quality Standard Widefield Like Fastest Best signal Smartest Line Linear unmixing Emission signal Speed A A AA Track 2 a N Cancel Fig 15 Proposals panel of the Smart Setup tool 11 2013 V_03 Motifs The feature Motifs is part of the Smart Setup panel Like in photography common imaging conditions with typical requirements also exist in laser scanning microscopy The different Motifs are presets of scanning parameters pixels size pinhole diameter scan speeds etc addressing such conditions Fig 16 Upon selecting one of these options by mouse click the selected icon will be highlighted If the motif Current is clicked the current set of scanning parameters will be left untouched e Pressing the Apply button applies the selected proposal in Smart Setup as well as the motif iw a P s T a Current Quality Standard Widefield Like Fig 16 The Motifs
4. 6 Show all mode A focus in the development of ZEN 2012 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 11 2013 V_03 5 Workspace zoom N Acquisition Mode Float Dock tool EC Plan Neofluar 10x 0_30 M27 Fig 7 ZEN Window Layout configuration More features of ZEN 2012 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 Acquisition Parameter to the right and a new tool column automatically opens Alternatively use the context menu Move Tool group 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 2012 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
5. 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 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 Acquisition Experiment Manager T tH wo E Smart Setup V Show all Tools Select all Unselect all AF O N EO Find Focus Set Exposure Continuous Snap Tracki vV Z Stack 21 Slices Time Series Bleaching vV Tile Scan 5x5 Tiles Positions Regions K Start Experiment 4h ah 4H h Setup Manager S 10W All 646 Acquisition Parameter o 10 Integration Fhoton Counting x 0 gt Show A i S s a CEPS ASEL 0 wld s 1 0 Multidimensional Acquisition moii eee Photon Counting offset i 0 000 0 E r E 1 00 20 11 2013 V_03 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 Set Exposure Live Continuous or Snap buttons to start the scanning procedure to acquire an image e Scanned shown in windows
6. images are separate e Click on the Stop button to stop the current scan procedure if necessary 5 Select Set Exposure for automatic pre adjustment of detector gain and offset Set Exposure Select Live for continuous fast scanning useful for finding and changing the focus 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 e In the View Dimensions View Option Control Block activate the Range indicator check box Fig 23 E gt Clicking on the right hand side of the S button leads to a list of colors 11 2013 V_O3 Dimensions Displa Single Channel Fig 22 40 Interpolation Range Indicator Quick Color Setup Reuse Y Crop Positions Stage Image Display Dimensions Display Zoom Tools Channels Fig 23 h 40 lt Interpolation ET E E Single Channel Range Indicator Quick Color Setup Reuse Crop Positions Stage View Dimensions Control Block 21 Fig 24 Image Display i Channels Tracks wf Tracki al 405 458 Display Fig 25 Channels tool 22 Channels DAPI Cy3 T PMT wf 486 4514 Select All Unselect All ir k G 561 594 635 a Show all E 0 9 1 2 0 2 0 0 P The scanned image appears in a false color presentation Fig 24 If the image is too b
7. run switch Fig 2 3 to run It takes about 50s until the laser has reached the set output power green LED provided the warm up time of 5 minutes is already completed e Adjust the required power level with the control knob Fig 2 4 default position should be 11 o clock O O 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 GO OOO OO GO O OOO O OOO Le La La La La La La La La La La La La La La La La GOGO OOO OOO OGO OO OOO OO GO OO OOO OOO OO OOO GGO OO GO CO OO OOO OOO O Fig 2 Power supply of Ar ML laser 2 11 2013 V_03 Starting the ZEN software ZEN 2012 e Double click the ZEN 2012 icon on the WINDOWS desktop to start the Carl Zeiss LSM software The ZEN Main Application window and the LSM 710 LSM 780 Startup window appear on the screen Fig 3 a ZEN 2012 Main Application window c LSM 710 LSM 780 Startup window Fig 3 ZEN Main Application window at Startup a and the LSM 710 LSM 780 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 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 hardw
8. LSM 5 format is the native Carl Zeiss LSM image data format and contains all available extra intormation 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 11 2013 V_03 Using FCS e Click the FCS button for Fluorescence Correlation Spectroscopy e Use the FCS tool groups Setup Navigation Multidimensional Acquisition in the Left Tool Area to acquire and analyze FCS data w ih oD T fa 0 Count rate xyz SCan Snap s Time Series Start Experiment v Positions LI L Sn R Iri le U R LA T Laser Light Path Acquisition Fit Correlator al K G ge Sample Carrier Focus Incubator Multidimensional Acquisition D Time Series ma Positions 1 Information On Experiment Fig 30 FCS Panel Setting a configuration e Open the Light Path tool to define the light path laser lines and pinhole position Depending on the system configuration the Light Path displays the selected track which is u
9. Microscopy from Carl Zeiss LSM 710 LSM 780 Systems and GonfoCor 3 LSM Software ZEN 2012 November 2013 We make it visible Page CONTENTS oee E 1 MOGUCOM oire E A E EE 1 Starting the System cesse esse esse eee eenn 2 Introduction to ZEN efficient navigation ccs s sees sees sc ee esec eee eee 5 Serung up The Ta e Lee T 10 Configuring the beam path and lasers sss sssss esse sees eenn 13 SCANNING AN TAG L 19 Storing and exporting image data sss sss esse sese ees enesenn 24 Ufa FE S eeeene tenet ttt meer NE nett rt eerie ret nets terre ter ett ert eerie rr et reer rT 25 Switching off the SV SCSI cesse esse ees eee eee eenn 34 Introduction This Quick Guide describes the basic operation of the LSM 710 LSM 780 LSM 710 NLO LSM 780 NLO and ConfoCor 3 Laser Scanning microscopes with the ZEN 2012 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 Axio Examiner 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 710 LSM 780 LSM 710 NL LSM 780 NLO and ConfoCor 3 Laser Scanning Microscope including its original acc
10. are 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 After 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 11 2013 V_03 3 A Application bar B Menu bar C Main toolbar D Left Tool Area E Center Screen Area hosts up to three Image containers F Right Tool Area G Status Area Fig 4 ZEN Main Application Window after Startup with empty image container RS 12 P 7 TT 13 1 Main tool tabs 4 Mutidimensional tool selection panel 7 Tool 10 Image tabs 13 View controls 2 Configurations 5 Starting multidimensional experiments 8 Status bar 11 Displayed image 3 Action btons 6 Tool group 9 View tabs 12 File handling area Fig 5 ZEN Main Application Window after Startup with several images loaded 11 2013 V_03 Introduction to ZEN efficient navigation The ZEN 2012 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 to
11. are presets for typical imaging tasks The selected motif is highlighted If the option Linear unmixing is selected the system is set in the lambda mode automatically O Pressing the Set Exposure button will then optimize the settings of the Gain Master and offset for the given laser power and pinhole size Further image optimization from this point can be done easily 11 2013 V_03 15 Simultaneous scanning of single double and triple labeling Advantage faster image acquisition Disadvantage potential 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 Light Path tool in the Setup Manager tool group and the Channels tool in the Acquisition Parameter tool group to access the hardware control window to set up the beam path The open Light Path is shown in Fig 17 Channel Acquisition Experiment Manager m R K Smart Setup vV Show all Tools New AF O cor Find Focus Set Exposure Continuous Snap vV Z Stack 21 Slices Time Series Bleaching v Tile Scan 5x5 Tiles v G se Use Dye Color Detector Range Regions wf DAPI Chi 415 5170nm af Cy Chs1 535 690nm Setup Manager Ch 415 735nm Reflection Acquisition Parameter r Plate Ai Visible light MBS 445 f Invisible ligh
12. available in the Light Path tool of the Setup Manager tool group Fig 17 Switch track every selection box 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 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 Tracks line m 2 Track buttons To change between the tracks settings click on the appropriate track button The selected track is highlighted The setting table for configuration will be shown behind Add Track button 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 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 configurat
13. ch 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 for visible and invisible light check box and setting of excitation intensities slider If necessary open the Laser Selection of the main dichroic beam splitter MBS for visible and invisible light from the 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 QUASAR detectors ChS1 8 transmission ChD for the scanning procedure and Color Detector Range Ch1 416 522nm ch51 535 690nm Ch 415 735nm Reflection Detection bands and laser lines display Fig 18 Save current Track configuration as Configuration DAFI Ok Cancel Fig 19 Track configurations functions 17 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
14. essories and compatible accessories from other manufacturers may only be used for the purposes and microscopy techniques described in this manual intended use n 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 11 2013 V_O3 1 Starting the system Switching on the LSM system e Switch on the main switch Fig 1 1 and the safety lock Fig 1 2 e When set to ON the power remote switch labeled System PC provides power to the computer This allows use of the computer and ZEN software offline After complete startup the computer Is ready for the components start 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 Fig 1 Power remote switch Switching on the X Cite 120 or the HBO 100 mercury lamp 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 Ar ML Laser e f the Ar ML laser is required switch it on via the toggle switch Fig 2 2 on the power supply and turn the key Fig 2 1 The laser is automatically kept in standby mode for 5 minutes to warm up e Set the idle
15. ezo 1 L Optimize Sectioning and Step Correction Fig 26 Z Stack tool 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 a Start Experiment Click on the Start Experiment button to start the recording of the Z Stack GS 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 be scanned 11 2013 V_O3 Start Experiment button is grayed out and only single images can 23 Storing and exporting image data Save image Fig 27 Save Image buttons in ZEN ave in u Image Data 7 d la as Name Date taken Tags Size Rating 5 Toxoplasma_ZStack_001 czi Open Microscopy Environment Tiff File ome tif Fig 28 Save as window Tagged Image File Full resolution image window single plane gt Full resolution image window single plane Select file name and save Fig 29 Export window 24 e Tosave your acquired or processed images click on the Save or Save As button in File menu or click the m button in the main toolbar Fig 27 1 or click on the E button at the bottom of the File Handling Area Fig 27 2 e The WINDOWS Save As window appears e Enter a file name and choose the appropriate image format Note the
16. ill open S There is no image database any more like in the earlier Zeiss LSM software versions Dimensions hand indicator New image Fig 8 New image document in the Open Images Areas 11 2013 V_03 7 Advanced data browsing is available through the ZEN File Browser Ctrl F or from the File menu The ZEN File Browser can be used like the WINDOWS program Tile browser Images can be opened by 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 My Documents My Pictures B Desktop Axiovision 4 6 Icons AT CR 3 E s i L 7 wes d LRA SN demo data 4D cells AlzheimerFRETFLIM AlzheimerFRETFLIMs alzheimers amp fromChris 3 Cha C9 S M 3 Channels 8 bit 3 Channels 6 bit B Nice Small LSM Demo Image 0 T 0 15 N 0 24 kB 0 30 MB l 0 57 1 1 MB Gif Movies B Riken Meta images Kogure Best mdb vonZIKUP ZIKUP_ImageData mdb JenaKultur JofBioTech_CallforPapers 9 l gt lt LSM 4 2 Icons Alzheimers4D Arabidopsisleafs autofluo ence bilecanaliculi bonemarrow Movies 3 Channels 8 bit 3 els 8 bi 3 i 3 Channels 8 3 Channels 68 bit 3 Channels bit l 0 14 MB 0 41 MB 1 0 55 MB 69 kB Music 0 60 MB 0 75 h 0 87 MB FSN 1 1 MB 0 75 MB narration zen temp install ZEN2007 AIM AIM45 Daten_Olaf gt a Documents and Settings Cc sinastro candidaalbicans centrin cerebralar
17. ion used Remove button The track marked in the List of Tracks panel is deleted 18 11 2013 V_03 Scanning an image e Select the Acquisition Mode tool from the Left Tool Area Fig 20 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 alpha Plan Apochromat 100x 1 46 Oil Dh Frame diz gt K Start Experiment n Setup Manager c l Acquisition Parameter y Scan Area e Use the Scan Speed slider in the Acquisition Mode tool Fig 20 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 e Select the dynamic range 8 or 12 Bit per pixel in the Bit Depth pull down in the Acquisition Mode tool Fig 20 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 measurements or when imaging low fluorescence intensities 11 2013 V_O3 19 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
18. 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 interface For a more detailed description of the functionality tor the ZEN 2012 software please refer to the User Manual that is provided with your system 6 11 2013 V_O3 6 To create a new image document in an empty image container click the Snap or the O Set Exposure button For an empty image document press the New O New or buttons The new document is immediately presented in the Documents panel of the Right Tool 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 st
19. n 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 S Depending on the microscope configuration settings must be done manually if necessary 11 2013 V_03 Configuring the beam path and lasers e Click the Acquisition button Acquisition se The tool Smart Setup is an intuitive user friendly interface which can be used for almost all standard applications It configures all the system hardware for a chosen set of dyes Smart Setup e Click on the Smart Setup button to open the smart setup window This window can be accessed any time from the software to change dye combinations e Click on the arrow in the dye list and simply choose the dye s you want to use in your experiment from the list dialogue In this dialogue the dyes can be also searched by typing the name in the search field Smart Setup VY Show All gis Dye Alexa Fluor 488 Sa Recent Alexa Fluor 633 Alexa Fluor 546 Alexa Fluor 546 Alexa Fluor 633 DAPI Alexa Fluor 488 Alexa Fluor 647 Cy5 TRITC Search GFP Current Sta Dyes EGFP hmGFP Fastest Best signal PA GFP nmixing SuperGlo GFP WEGFP WTGFP
20. ols 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 dual 20 TFT setups x Time Series oo Show all mode active E owai A Cycles Interval Interval Time Interval Time interval Time Trigger In Trigger Out 100 0 msec Triggeri v Trigger2 v Show all mode inactive Time Series Cycles a Trigger In Trigger Out Interval None lt None Y Fig
21. ountrate Correlation Co sper Amplitude Triplet state Triplet state omponent1 Component Tra e Ni umber particles Fraction Relaxation time Fra ction Diffusion time ee J W 32 2 24 2010 12 22 13 average Rh6G 102 842 19 558 V 33 24242010 12 2213 average Cross correlation Ch2 vs Ch 2885 Fig 37 FCS Correlation diagram You have the following function elements Activate the Correlation panel to display measured data Fig 37 P Activate the Coincidence panel to display a scatter plot of the two channels cross Coincidence correlation only Activate the Fit panel to select a model and fit the data to it Activate the Print panel to set up a connected printer and print a hardcopy of the data Activate the Information panel to give the data a name input any comments and view Information the Meta data Save the data by clicking the File button in the menu bar 11 2013 V_03 31 E 0 0001 0 004 Lag Tire 3 Fitrange 0 600 psto 3 566 Correlation Fit deviation mia 0 10 0 08 0 06 0 04 0 02 0 00 0 07 0 04 0 06 1e 006 0 00001 0 0001 0 001 0 01 Lag Time s Mumber Date Time Repetition Channel Countrate Correlation Counts per Amplitude A kHz molecule Murmber particles G RAGS 0 SSF Cross correlation Ch2 vs Cn Fig 38 FCS Fit diagram 32 11 2013 V_03 You have the following options f Correlation Parameter a LLS Fit All To Method 11 2013 V_O3 Ac
22. ove into the beam path Focusing the microscope for transmitted light e Open the graphical pop up menu by clicking on the Transmitted Light icon Fig 12 e Click on the On button Set the intensity of the Halogen lamp using the slider e Clicking outside the pop up control closes it e Place specimen on microscope stage The cover slip must be facing the objective lens Re member the immersion medium if the objective chosen requires it 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 11 2013 V_O3 Ocular Fig 11 None EC Plan Neofluar 100 3 Aperture 0 16 HF Microscope Control window e g Axio Imager Z2 11 Transmitted light control Reflector Reflected light shutter Plan Apochromat 63x 1 40 Oil DIC L M27 Reflected light source N X Cite 120 or Off 0 Closed HBO 100 Fig 12 Microscope Control window with Transmitted Light pop up menu Configuration Assign Fig 13 Configuration panel 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 ca
23. right 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 25 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 25 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 25 e Reduce the Gain Master until the red pixels only just disappear 11 2013 V_O3 Scanning a Z Stack Select Z Stack L 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 Continuous Click on the button in the Action button area A continuous XY scan of the set focus position will be performed Use the focus drive of the microscope to focus on the upper position of the specimen area where the Z Stack is to start Click on the Set First button to set the upper 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 P Show all E First Last Set Last E 10 6 936 z 62 426 ym Interval Slice Set First 31 21 wf Use Pi
24. rmyloidangio Administrator Rz 3 8 b 3 Channels 8 bi 3 Cha bi 3 Cha S 3 Channels 8 bit All Users MB 0 WB l D 0 B tu 0 52 MB 0 57 MB 0 75 MB 58h 0 56 MB 0 87 MB Default User HPPET M105E Application Data B Desktop Axiovision 4 6 Icons amp demo data 4D cells i fromChris cervicalsmear cy 2 S cysticproximaltubules Daphn DAPI amp Nice Small LSM D 3 Channels 8 bit 3 Channe bi 3 nels 8 bit 3 Channels 8 bit 3 Channels 8 bit 3 Channels 8 bit l l MB 0 0 11 MB B 48 kB Gif Movies 0 58 MB 0 75 MB 0 75 MB 0 87 MB 0 44 MB Imaaes S L Cut Sl Copy Fig 9 File Browser 8 11 2013 V_O3 Turning on the lasers ZEN 2012 operates all lasers automatically Whenever they are used manually or by the Smart Setup function the lasers are turned on automatically The Laser Life Extender function of the software shuts all lasers off if ZEN is not used for more than 15 minutes To manually switch lasers on or off e Click the Show all tools tickbox and open the Laser tool All available lasers can be operated within this tool Fig 10 Acquisition K Smart Setup vV Show all Tools New AF O l cJ Find Focus Set Exposure Continuous Snap vV Z Stack 21 Slices Time Series Bleaching vV Tile Scan 5x5 Tiles Positions Regions Setup Manager Laser Light Path Acquisition Parameter Acquisition Mode Channels t Focus Pi Focus Devices and Strategy Stage L Shuttle and Find Incubator
25. sed for the FCS procedure The pinhole position and size can be set by pressing the Adjust pinhole button The laser line and power can be set in the Visible light Invisible light drop down menu by pressing the respective laser icon see Fig 31 11 2013 V_03 25 2 Channel Module 3 Channel Module c Visible light KTN Visible light c Invisible light Invisible light m Ip C2 i GaAsPl BP 530 610 IR W Chi gt ch ee e Ye BP 500 550 w GaAsP2 gt GaAsPl wf Ga sP1l gt GaAsP2 La L k LP 655 Adjust pinhole w Ll wD Channel Module 32 Channel Module NGOO ooooooo CUR Visible light UES Invisible light Auto Correlation Detector dU J chs 49 630nm vf chS2 638 69Cnm Pmt 754 758 nm lt NDD R3 T NDD R4 Cross Correlation Detector A Detector B af Che ChS7 F chs2 Chs1 MBS 488 633 Ld ml d E Visible light gt c gt Plate 4 _ oe WNDDOT3 9 NDDT4 L Invisible light Stage Focus Incubato Cross Correlation Detector A Detector B wf NDD R3 NOD R4 f NDD R4 NOD RS Adjust pinhole Collimators Fig 31 Light path tool of a LSM 780 system with the 2 Channel mode ConfoCor 3 above left 2 Channel mode BiG above right 32 channel mode Quasar below left and Channel mode NDD below right 11 2013 V_O3 You can change the settings of this panel using the following function elements Activation deac
26. t NonelsM 4 F F ps Show a i A j 4 Stage Focus T PMT gt Multidimensional Acquisition Ratio 16 11 2013 V_03 Settings for track configuration in Channel mode e Select Channel mode if necessary Fig 17 Control tool see above relevant list box gt assigning a color to the channel Select the appropriate filters and activate the channels Fig 17 Click the Laser icon to select the laser lines and set the attenuation values transmission in in the displayed window Fig 17 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 Fig 18 The Detection Bands amp Laser Lines are displayed in a spectral panel Fig 18 to visualize the activated laser lines for excitation vertical lines and activated detection channels colored horizontal bars For storing a new track configuration open the Channels tool in the Acquisition Parameter tool group click S and enter a desired name in the first line of the list box Fig 19 then click Ok to store the configuration For loading an existing configuration click ow then select it from the list box For deleting an existing configuration click ES then select it from the list box and confirm the deletion with Ok 11 2013 V_03 The Light Path tool displays the selected track configuration whi
27. tivate the Fit panel to display fitted data see Fig 38 Set the red and blue bars to define the start and end points of the curve fit window Load a predefined model from the Model drop down menu You can assemble a model by pressing the Model tool in the FCS tool group Define the conditions of the fit by activating deactivating terms setting the type of a parameter fixed free or start value defining limits and globally link parameters in the Model table Pressing the Fit button will fit the current loaded correlation functions to the defined model The fitted data will be displayed in the Model and Result tables Pressing the Fit all button will take all ticked channels and fit them according to the selected model Pressing the Undo button will undo the last operation or previous ones as well if the button Is pressed repeatedly Pressing Redo will redo the last cancelled operation or previous ones if the button is pressed repeatedly Pressing the To Method button will write back the settings to the method If the method is stored the settings will be active when the method is selected the next time 33 Switching off the system 34 Click on the File button in the menu bar and then click on the Exit button to leave the ZEN 2012 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 Switch off the Ar ML laser
28. tivation of the excitation wavelengths check box and setting of excitation intensities slider Open the Laser Control tool by clicking the Laser icon Visible light Invisible light Selection of the main dichroic beam splitter MBS or secondary dichroic beam splitter SBS position ConfoCor 3 only through selection from the relevant list box Selection of a block filter ConfoCor 3 only through selection from the relevant list N aa box Selection of an emission filter ConfoCor 3 only through selection from the relevant i list box BP 330 610 IR Activation deactivation via check box of the selected channel shown for the ConfoCor 3 and Quasar Auto Comelaton Detector w Chs1 Adjust pinhole and collimator e Set the pinhole Diameter via slider or input box not available tor NDD marre e Press the Count rate button to open the Real Time display window for the detector Count rate in all active channels Adjust the laser power in the Laser Control panel to obtain a satistactory count rate 1AU 1 00AU e Press Auto adjust buttons to align the pinhole for each newly defined beam path After adjusting the sample carrier align the pinhole automatically in X and Y by conducting a coarse first and then a fine alignment Fig 32 Pinhole adjustment 11 2013 V_O3 27 Use the Fit tool to define model equations to which measured data can be fitted by pressing the Chs2 Chs1 Define bu
29. tton Define raat There are three options to define a model Correlation assemble a correlation model from predefined equations which will be Correlation fitted analytically Offset PCH assemble a photon counting histogram model which will be fitted numerically Background Formula program a user defined model s Amplitud i ettings i j i Amplitude saanu equation which can be fitted analytically Antibunching w Triplet Settings Rotation wf Translation Flow Stretched Exponential Gd lt 1 A G Gy Cancel Fig 33 Fit tool above and Define Model below windows 28 11 2013 V_03 e Open the Acquisition tool The panel of the Acquisition tool displays the selected measurement conditions used for the FCS experiment Enter the Measure Time Repetitions and Bleach Time into the corresponding input boxes and select the colors for the correlation functions Activate the required lasers and set the laser power e Open the Time Series tool Define a kinetic procedure by entering the cycle Number the Point to point time distance between measurements and the Shape in the corresponding input boxes e Open the Positions tool Select the carrier and the sample or laser position by the stage or by the scanners 11 2013 V_O3 al 405 440 458 488 4514 561 594 635 1 000 Pupil filling Pinhole Z AU 1 00 AU Fig 34 Acquisition tool Fig 35 Time Series tool
30. with first the idle run switch Fig 2 3 and second the key switch Fig 2 1 then wait until the fan of the Argon laser has switched off Don t switch off the main power yet On the power remote switch 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 Ar ML laser of by the main power switch on the power supply Fig 2 2 11 2013 V_O3
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