Carl Zeiss LSM 710 NLO User's Manual
Contents
1. 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 E Light Path Acquisition Color Detector Range chi 409 484 nm h51 499 348 nm Che 296 696 nm Reflection d Find Continuous MBS 488 594 a mh Visible light j Setup Manager Laser es O m Imaging Setup MBS 405 d E Light Path et Online Acquisition i me None Invisible light j Multidimensional Acquisition D ZStack Bleaching O Time Series Tile Scan i Information On Experiment Ratio Fig 14 Light Path tool for a single track LSM 05 2008 13 Settings for track configuration in Channel Mode 4 Imaging Setup e Select Channel Mode if necessary Fig 15 Seale nit ee ES e Click on the LSM tab Fig 14 nai ic iat me The Light Path tool displays the selected track Switch track every Frame configuration which is used for the scan procedure So e You can change the settings of this panel using the ch2 following function elements 500 600 700 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 s2. 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 tor a 30 TFT monitor but can also be used with dual 20 TFT setups w amp Time Series Pro Mode ie Interval Cycle Delay Time Cycle Delay Time experiment 1 Trigge Trigger2 v Basic Mode w Time Series 3 Marker Cycles za Marker not defined Interval ED anm CD CD Fig 6 Basic and Pro Mode A focus in the development of ZEN 2008 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 6 05 2008 The Pro Basic concept ensures that tool panels are never more complex than needed In Basic Mode the most commonly used tools are displayed For each tool the user can activate Pro Mode to display and use additional functionality Fig 6 Workspace Zoom w iz Light Path Acquisition Fig 7 ZEN Window Layout configuration More features of ZEN 2008 include e The user can add more columns 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
3. 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 Channel Chi Pinhole Find 0 00 Airy Units 9 8 pm section Continuous Master Gain Setup Manager Laser Imaging Setup E Light Path Digital Offset Online Acquisition Digital Gain Q Acquisition Mode nian Track Track 1 wi Multidimensional Acquisition 40 5 4 5A 450 5 q1 4 56 q 594 6 3 3 ZStack Bleaching l Time Series 405 nm Tile Scan i Information On Experiment Fig 19 Channels tool 05 2008 17 Image acquisition Image optimization View Dimensions Z Position 1 Zoom Channel s Merged Chi Ch2 l I R Other Fig 21 View Dimensions Control Block 18 Once you ha
4. LP 470 O Laser Lines Pinhole Collimator Fig 29 The ConfoCor 3 Measure Tool System Configuration 22 05 2008 You can change the settings of this panel using the following function elements Pinhole 05 2008 70 00 Count rate Adjust Pinhole Activation deactivation of the excitation wavelengths check box and setting of excitation intensities slider Open the Laser Control tool via the Laser icon Selection of the main dichroic beam splitter HFT or secondary dichroic beam splitter NFT position through selection from the relevant list box Selection of a block filter 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 Setting of the Pinhole diameter via slider or input box 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 satisfactory count rate Press Adjust Pinhole to align the pinhole for each newly defined beam path After adjusting the sample carrier align the pinhole in x and y by first conducting a coarse and then a fine alignment 23 Starting a measurement f Measure ww E Measure e Open the Measure toolbar to i Acquisition access experiment parameter Times Calculated Duration 10 s control
5. 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 Master Gain until the red pixels only just disappear 05 2008 Fig 22 Image Display Channels Channel Chi Pinhole 0 00 Airy Units 9 8 pm section Master Gain gt Digital Offset Digital Gain Track Track 1 v 405 456 466 3174 367 394 635 405 nm Fig 23 Channels tool 19 Scanning a Z stack Z Stack 7 Mode e First Last Center Number of Slices Interval Keep Interval Slice Optimal Interval 3 08 ym 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 SELES button in the Action Button area A continuous XY scan of the set focus position will Set Last 20 241 be performed Set First 25 695 Range 45 94 um Focus Position 2 73 ym Z Stack Navigation Move to id Last Center First Fig 24 Z Stack tool 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 Click on the be button to set number of slic
6. will never be over written and a new scan will then automatically create a new image document Alternatively you can create a new empty image document with the New button in the Action Button area or the New function in the File Menu 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 Note There is no image database any more like in the earlier Zeiss LSM software versions New Image mmo I Fig 8 New image document in the Open Images Ares 8 05 2008 Advanced data browsing is available through the 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 Fig 9 05 2008 My Documents My Pictures Desktop Axiovision 4 6 Icons demo data 4D cells 2nd fromChris 3 Channels 0 33 MB 0 75 MB Nice Small LSM Demo Image Gif Movies aru Riken Meta images Kogure Best mdb vonZIKUP ZIKUP_ImageData mdb JenaKultur JofBioTech_CallforPapers x LSM 4 2 Icons Alzheimers4D Movies 3 Channels 8 bit 0 14 MB Music 0 60 MB narration zen temp install ZEN2007 AIM AIM45 Daten_Olaf Docu
7. 2 Find Continuous Optimal Setup Manager O Laser amp Imaging Setup E Light Path Speed Online Acquisition Pixel Dwell 1 60 psec Scan Time 3 93 sec Acquisition Mode 4 Channels O Focus gt Stage 9 Regions Averaging Multidimensional Acquisition N umber p Bit Depth B Bit D ZStack Bleaching Time Series A a 7 Tile Scan i Information On Experiment Fig 18 Acquisition Mode tool Scan Area Note When using an Axioskop 2 FS MOT make sure you set the correct objective manually in the Acquisition Mode tool This ensures correct calibration calculation of pinhole and Z stack optimization etc The Axioskop 2 FS MOT does not automatically detect the objective lens 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 measurements or when imaging low fluorescence intensities 16 05 2008 Setting scan averaging
8. 2 average Cross correlation Che vs 1431 050 m Options pro Correlation Diagrams Options Correlation Diagrams Diagrams O Display Options Fig 31 FCS Correlation diagram You have the following function elements Fcs Correlation Activate the FCS Correlation panel to display measured data Fig 31 Press the View Options button to define the graph you want to display Press the Count rate button to display the count rate trace Press the Correlation button to display the correlation function Press the Photon counting histogram button to display the photon distribution per time unit 26 05 2008 Press the Data Options to handle your data Press the Save Data button to open the Save window You can save the whole data set in an ANSI text format Optionally you can save the raw data trace if that option was set in the FCS Options Pressing the Reuse button will set the system configuration to exactly the same values as used in the experiment Pressing the Reload button will open the current measurement if stored raw data are available This allows you to alter the parameters of your mathematical calculations I L L i L pi The acquired FCS data is analyzed in the Fit display of the FCS diagram see figure Fig 32 OC example file Model model name Channel Auto correlation Ch2 Parameter Value Lower limit Upper limit Link Humber particles 1 644 0 000 1e 010 Amplitude Geomet
9. Fit all button will take all ticked channels and fit them according to the chosen model Pressing the Undo button will cancel 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 Write 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 05 2008 Switching off the system e Click on the File button in the Main Menu bar and then click on the Exit button to leave the ZEN 2008 software e lf any lasers are still running you should shut them off now in the pop up window indicating the lasers still in use e Shut down the computer e Switch off the Ar ML laser with 1 the standby switch Fig 2 3 and 2 the main switch Fig 2 2 and wait until the fan of the Argon laser has switched off Don t turn the key switch yet e On the power remote switch turn off the Components switch and the System PC switch Fig 1 e Switch off the X Cite 120 lamp or the HBO 100 mercury burner e Switch off the Ar ML laser of by the main key switch on the power supply Fig 2 1 05 2008 29
10. Main Application window at Startup a and the LSM 710 Startup window b and c In the small startup window choose either to start the system online 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 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 After Startup the ZEN Main Application Window Fig 4 and Fig 5 opens To benefit trom all of Zen s features run the window in its Tull screen mode 4 05 2008 Application Bar Menu Bar Main Toolbar Left Tool Area Center Screen Area hosts up to 3 Image Containers Right Tool Area Status Area Fig 4 ZEN Main Application Window after Startup with empty image container 1 Tabs to switch between tool types 6 View Tabs 2 Action Bu
11. Microscopy from Carl Zeiss LSM 710 LSM 710 NLO and GonfoCor 3 LSM Software ZEN 2008 August 2008 We make it visible Page NS ta stro ete EEE E 1 BAEC CEO ata bt ces ence tapes iste neers sti ements E eee mae et cee E ne smeeteaeee 2 Starting the System cases cette ee ts ase tn tes ne tse me te ea ome one eee pet enee ees ween 3 Introduction to ZEN Efficient Navigation ccccscceeeeesseeeeeeeeeeeeaneessaeseeeseaseessaaneessonnaes 6 Setting up the WCU OSC Cae e tects ate re vacated en ctppc ee ined ene nate sa vacduniiseceeeimucevandcueeeeaie 11 Configuring the beam path and lasers cccccseeeeeseeeeeeseeneeeseaseeessaeseeesaeeesseaseesseaseeessees 13 Scanning an MMM C ocectec eee tire ocavecie notion steceanecounecowetiocateacsmaocsrecemetaveiaandcues ward etaueiaete end sande 16 Storing and exporting image data cccceeeeceeeeeeeeeneeeeeneeeeeeeeseeeessaeeseeeeseeneessonnessansessnaes 21 Using the ConfoCor 3 module cotati ese tec eet teeeiee tee riect eet ee eee eee 22 Switching Bee Wik a ek C 9 eee eee eer eee ee eee eee eee ee eee 29 05 2008 1 Introduction This LSM 710 LSM 710 NLO and ConfoCor 3 Quick Guide describes the basic operation of the LSM 710 LSM 710 NLO and ConfoCor 3 Laser Scanning microscopes with the ZEN 2008 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 s
12. amples 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 710 NLO and ConfoCor 3 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 2 05 2008 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 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 e Switch on the main switch of the X Cite 120 HBO 100 lamp for reflected l
13. and presented as the front tab in this tool 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 Use the focusing drive of the microscope to focus the object plane Select specimen detail by moving the stage in X and Y using the XY stage fine motion control 05 2008 O E Light Path Ocular Configuration ofso Closed 100 63 64 EC Plan Neofluar 10x 0 30M27 lt Aperture 0 3911 JO On 50 Open Fig 11 Microscope Control window e g Axio Imager Z1 amp EF Light Path Reflected Light assis Source X Cite 120 Configuration or HBO 100 C C A a aN Reflected Light ue Closed 100 63 Shutter i EC Plan Neofluar amp 10x 0 30 M27 Reflector Focus a Transmitted Light Aperture 0 3911 a Control BF Ad A ilsa 4961 Fig 12 Microscope Control window with Transmitted Light pop
14. ee above Selection of the main dichroic beam splitter MBS 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 QUASAR detectors ChS1 8 transmission ChD for the scanning procedure and assigning a color to the channel e Select the appropriate filters and activate the channels e Click the Laser icon to select the laser lines and set the attenuation values transmission in in the displayed window e For the configuration of the beam path please refer to the application specitic configurations depending on the used dyes and markers and the existing instrument configuration e Inthe Imaging Setup tool the Detection Bands amp Laser Lines are displayed in a spectral panel Fig 16 to visualize the activated laser lines for excitation vertical lines and activated detection channels colored horizontal bars Fig 16 Detection Bands amp Laser Lines display 14 05 2008 e For storing a new configuration enter a Imaging Setup desired name in the first line of the Cria kA C fi li k B Alexa 488 Cy3 seq onfigurations list box Fig 17 and clic Mode simultaneous Alexa 488 Cy3 sim Store Switch track every duo seq p gi i i NLO 1 e For loading an existing configuration select it V tag Me ose 1 from the list box and click on the ca Load gg a
15. es to match the optimal Z interval for the given stack size objective lens and the pinhole diameter Make sure that the activation tickbox is set Click on the Start button to start the recording of the Z Stack Note When ETOD is not activated the respective tool and its set parameters are not included in the multidimensional image acquisition If no multidimensional tool is activated the i Start button is grayed out and only single images can be scanned 20 05 2008 Storing and exporting image data e To save your acquired or processed images click on the Save or Save As button in File Menu Fig 25 1 click on the EJ button at the bottom of the File Handling Area Fig 25 2 or click the B button in the Main Toolbar Fig 25 3 Fig 25 Save Image buttons in ZEN e The WINDOWS Save As window appears Speichern unter e Enter a file name and choose the appropriate Speicher 64 Desktop a image format Note the LSM 5 format is the Qeigene Dateien demo data ti C 7 LSM d t f t d H Arbeitsplatz 9 Jenakultur Nd Ive ar EISS Image Ud a orma an hetzwerkumgebung C JofBioTech CallforPapers contains all available extra information and ciscoSecure Authentication Agent LSM 4 2 Icons i i Btik Movies Movies hardware settings of your experiment a A e Click on the SAVE button k If you close an image which has not been saved a Dateiname ug _Speichen pop up window will ask you if you
16. ight 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 turn the key Fig 2 1 and switch it on via the toggle switch Fig 2 2 on the power supply e After about 10 min switch trom standby to run by the switch Fig 2 3 e Adjust the required power level with the control knob Fig 2 4 default position should be 11 O clock 05 2008 Fig 1 Fig 2 Power remote switch Oo Oo Oo O Oo Oo 0 O 0 Oo O O 0 0 Oo O 0 VCSSSSSogogogggssggsgssa SSSSSSSoggogogesgsssss QSSSSSSSoggggggsgsss QSSSSSoSoogoggsgsssssss VSSSsogogogogeggssssss QSSSSsgoogogoggssggsgss Power supply of Ar ML laser Starting the ZEN software amp e Double click the ZEN 2008 icon on the WINDOWS desktop to start the Carl Zeiss LSM software EEN eUS The ZEN Main Application Window and the LSM 710 Startup window appear on the screen Fig 3 Login ZEN 2008 Laser Scanning Microscope LSM 710 Boot Status Start System Image Processing Login ZEN 2008 Laser Scanning Microscope LSM 710 Boot Status Login ZEN 2008 Laser Scanning Microscope LSM 710 Boot Status CP Module Loaders CP Interfaces CP Realtime Controllers CP RtBoards a ZEN 2008 Main Application window Hardware configuration database ee CP RtBoards Start System Offline Demo Cancel c LSM 710 Startup window Fig 3 ZEN
17. l 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 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 Add Track button The track marked in the List of Tracks panel is deleted _ Remove button A click on this arrow button will move the selected track highlighted in light grey one position upwards in the list box A click on this arrow button will move the selected track highlighted in light grey one position downwards in the list box 05 2008 15 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 The number of pixels influences the image resolution w Acquisition Mode Objective Plan Apochromat 63x 1 4 Oi DIC Scan Mode Frame Frame Size X 512 x Y 51
18. ments and Settings Administrator 3 Channels 8 bit All Users 0 29 MB 0 52 M Default User mo HPPET MiOSE Application Data a Desktop Axiovision 4 6 Icons e eo amp demo data ea es 4D cells cervicalsmear fromChris amp Nice Small LSM D 3 Channels 8 bit 0 iB Gif Movies 0 58 MB File Browser 3rd 3 Channels 8 0 19 MB 0 75 MB ciliate 3 Channels 8 bit MB 0 56 MB candidaalbicans cy sticrmousekidney 3 Channels 65 bit 0 75 MB AlzheimerF RETFLIM 3 Channels 68 bit 0 vB 0 13 MB 0 58 MB proximaltubules 3 Channels 65 bit 0 11 MB 0 75 MB pe 5 gt 4 fa en me Ps he gt si ry 4 i Rx es we AlzheimerFRETFLIMs alzheimers 3 Channels 83 bit 0 30 MB 1 1 MB bonemarrow 3 Channels 8 bit 59 kA 0 75 MB 0 87 MB Daphn DAPI 3 Channels 8 bit 3 Channels 8 bit 48 kB 0 44 MB Turning on the lasers e Open the Laser Tool in the left tool area always the first in the list and activate the lasers you need for your experiment Fig 10 Remember the argon multi line laser has to first be put to standby for a 5 minute warm up before it changes to on e Zeiss recommends operating the Argon multi line Laser at a tube current of about 6 A 50 output This is the best compromise between laser stability oower and laser life time tube current control can be found in Pro Mode 10 Find Continuous Setup Manager O Laser a
19. mp Imaging Setup E Light Path Online Acquisition 4 Acquisition Mode Channels OD Focus DS Stage Regions Multidimensional Acquisition Q ZStack Bleaching O 2 Time Series Tile Scan i Information On Experiment Fig 10 gt Laser Control tool in Pro Mode DPSS 561 10 Chameleon HeNe633 Laser Information Maximum Power Wavelength Status Tube Current Output 30 0 mW 456 477 466 514 nm Warming up 6 0 A 05 2008 Setting up the microscope Changing between direct observation camera detection and laser scanning mode The Ocular Camera and LSM buttons switch the beam path and indicate which beam path is currently in use for the microscope c7 Ocular gt Qoular Workspace Zoom Workspace Zoom are blocked r Camera system Camera Setting up the microscope and storing settings e Click on the Ocular button to change the microscope beam path for direct observation via the eyepieces of the binocular tube lasers e Click on the Camera button to change the beam path of the microscope for the port where the camera is attached for camera based image acquisition e Click on the LSM button to set the beam path for the LSM 710 Click on the Ocular button for direct observation Then choose the Light Path tool from the Left Tool Area Since the Ocular button has been chosen before the Oculars tab is pre selected
20. new tool column automatically opens To detach a tool click on the little icon on the very right end of the blue tool header bar Fig 7 e Another unique feature in Imaging Software is the scalable ZEN interface This Workspace Zoom allows adjustment of the ZEN 2008 window size and fonts to the situational needs or your personal preferences Fig 7 e 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 e 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 2008 software please refer to the User Manual that is provided with your system 05 2008 7 To create a new image document in an empty image container click the Single a or the Start 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
21. ric correction 1 000 0 000 Je o10 Triplet state Fraction 14 559 0 000 Fics Fit Relaxation time us 36 469 0 000 Component 1 Fraction 0 000 Diffusion time us 248 266 0 000 1e 010 1e 006 O 00001 0 0001 0 001 oO 01 Time 5 Structural parameter 49 443 0 000 je 010 G tt J Fit dewiation 0 10 0 00 12 0068 0 00001 0 0001 oO 001 0 01 Time 3 Fit Fit All Undo Number Repetition Channel Correlation Amplitude Counts per Component 1 Number particles molecule Difusion time Auto correlation Chi 240 hE Cross correlation Ch vs i 1 469 142 210 638 Options Fit Diagrams Options Fit Diagrams Fig 32 FCS Fit diagram 05 2008 27 You have the following options Activate the FCS Fit panel to display fitted data Fig 32 Model model name Parameter Ri Undo 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 ConfoCor 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
22. s e Select the Acquisition controls 00 in the Measure tool Kinetics Positions The Light Path and Pinhole panels of the Measure graian window display the selected track configuration which is i eee used for the FCS procedure and the pinhole size see Fig 30 Scanner XY Stage Vv x Position 0 00 y Position Step Size um 1 Speed Settings Fig 30 The ConfoCor 3 Measure Tool Acquisition The Times Kinetics and Position panels of the Acquisition window display the selected measurement conditions and the positions which are used for the FCS experiment You can change the settings of this panel using the following function elements Times Enter the bleach time measurement time and repetition number in the corresponding input boxes Kinetics Activate deactivate a kinetic procedure by ticking the Kinetics check box Enter the time distance between measurements and the cycle number in the corresponding input boxes Positions Select a sample carrier position and be sure that the focus is within the sample volume solution or location in an LSM image eT ae 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 Ht NO 4 05 2008 Press the New button to open a new FCS diagram into an image container If a meas
23. s omer button test meta a l o z stack config e For deleting an existing configuration select it in p Fig 17 the list box and click on Delete 3 Track Configurations window 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 8 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 laser line intensity and the Amplifier Offset are switched but no other hardware components The tracks are al
24. ttons 7 Image Tabs 3 Tool Group 8 Displayed Image 4 Tool 9 File Handling Area 5 Status Bar 10 View Controls Fig 5 ZEN Main Application Window after Startup with several images loaded 05 2008 Introduction to ZEN Efficient Navigation ZEN Efficient Navigation is the new software for the LSM Systems from Carl Zeiss With the launch of this software in 2007 Carl Zeiss sets new standards in application friendly software for Laser Scanning Microscopy The ZEN 2008 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 image acquisition image processing and system maintenance easily accessible via 3 Main Tabs Fig 5 1 All functions needed to control the microscope and to acquire images are bundled in 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 Blocks below each image File management and data handling tools are found in the Right Tool Area see Fig 4 and Fig
25. up menu w E Light Path Ocular Configuration Fig 13 Saving Microscope configurations 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 Ea load button The current config can be deleted by pressing the delete button In Pro Mode these configurations can be assigned to buttons that are easier to press Note Depending on the microscope configu ration settings must be done manually if necessary 05 2008 Configuring the beam path and lasers e Click on the LSM button Workspace Zomm Olar Camera Choosing 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
26. urement is triggered all data are displayed in that window if highlighted Press the Start button to trigger a measurement All defined positions will be approached consecutively Press the Single button to trigger one measurement at the highlighted or first defined position Press the Stop button to end a measurement All data accumulated so far will be available and can be stored 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 XY Stage if positioning should be accomplished by the motorized stage Press Scanner if the positioning should be accomplished by the scanning mirrors After measurement completion the data is displayed in the FCS Correlation diagram within an Image Container see Fig 31 05 2008 25 Lx CC example file Fes Correlation Count rate kHz 30 0 001 0 01 ime 54 O 00010 0 0003 0O00 o003 ODO 003 O10 5 Pulse distance ms Counts binning time 3 28 ms Correlation Amplitude Counts per Countrate Component Number particles molecule kHz Difusion time kHz Hs 2 Auto correlation Chi 1 695 1 438 3 427 aaea a 1 744 1 345 pa Cross correlation Ch ws 1 475 0 915 average Auto correlation Ch 1 675 1 461 3 332 4 936 average Auto correlation Che 1 606 1 644 4 2b 7 007
27. ve set up your parameter as defined in the above section you can acquire a frame image of your specimen e Use one of the Find Fast Continuous or Single 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 Find Select Find for automatic pre adjustment of detector gain and offset Fast Select Fast for continuous fast scanning useful for finding and changing the focus Continuous Select Continuous for continuous scanning with the selected scan speed Select Single 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 click inside the color field in the button under the channel button Fig 21 Note Clicking on the right hand side of the button leads to a list of colors 05 2008 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 Detector Gain high e When the image is saturated reduce AOTF transmission in the Laser control section of the Channels Tool Fig 23 using the slider
28. want to save it Dateituo 3 Abbrechen Choosing yes will lead you to the WINDOWS Save As window Fig 26 Save as window To export image display data a single optical section in raw data format or the contents of the TETTERE image display window including analysis and CETERAN REEE E overlays choose Export from the File Menu In the Export window you can select from a number Contents of image window single plane of options and proceed to the WINDOWS Save As sein aa E window to save the exported data to disk Seinen cehabincel Select file name and save Fig 27 Export window 05 2008 21 Using the ConfoCor 3 module e Click on the LSM button Workspace Zomm e Use the ConfoCor 3 Tool Group in the Left Tool Area to acquire and i avi pa analyze FCS data ContoCor Count rate e 8 z 2 5 5 S 6 S URK b gt i Bi PERS 8 Ww i g o g gt W ee gt y xyz Scan 9 ooo a E e fi m F SISTSATSA w ee ee a Fig 28 ConfoCor 3 Tool Group Fiz e Open the Measure toolbar to System Configuration access experiment parameter Light Path controls e Select the System Configuration controls in the gt Measure tool The Light Path and Pinhole panels of the Measure window display the selected track configuration which is used for the FCS procedure and the pinhole size see Fig 29 V AC Ch V CcCCh1 gt Ch2 VY CC Ch2 gt Ch1 o
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