Operating Manual LSM 710 and ConfoCor 3
Contents
1. 6 7 8 a ZEN 2009 Fie Acquisition Maintain Macro Tools View Viindow Help Workspace Zoom s Workspace Configuration 1 i Smart Setup Show manual tools PA O n a 2 Live O75MB o 2 Steck 21 Siice s T S V Time Ser 10 Image s A C V Bleaching t iterations l 0 75 MB Regions 2 on Onine Acquistion gt _ A AlzheimerFRETFUM jpg 3 Mode 4 c t Focus a 0 87 MB Multidimensional Ac aquisitior c 043 MB 9 yD cement 0 00 pm SN 5 j 4 interval Si P r 7 ae Set First 10 000 Sem autofluorescence jpg Time Series i Information On Experiment 10 5 1 Tabs to switch between the tool types 6 View tabs 2 Start buttons 7 Image tabs 3 Tool group 8 Displayed image 4 Tool 9 File handling area 5 Status bar 10 View controls Fig 5 ZEN Main Application Window after Startup with several images loaded 04 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 work2. 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 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 06 20 20 20 20 20 20 20 20 20 20 20 20 20 ae Co QSSSSSSSSSgogggsssa VSSSSooggogegogsgsss e Adjust the required power level with the control knob Fig 2 4 default position should be 11 o clock VSSSSSSogogoggggssa QSSOSSSSSSoggogegsesss Fig 2 Power supply of Ar ML laser 2 04 2009 Starting the ZEN software 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 710 Startup window appear on the screen Fig 3 Login ZEN 2009 Laser Scanning Microscope LSM 710 gt Boot Status Start System Image Processing Login ZEN 2009 Laser Scanning Microscope LSM 710 Boot Status Helbling Stops Triggers Monitordiodes RtCamerss a ZEN 2009 Main Application window Pamsimas Hardware configuration database Potential Objective Lenses S
3. 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 17 Fig 18 and Fig 19 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 all matched to the current track with regard to emission filter dichroic beam splitter setting of Detect
4. Reflected light source down list and pressing the load button The X Cite 120 or current config can be deleted by pressing the HBO 100 delete button Fig 12 Microscope Control window with Transmitted Light pop up menu These configurations can be assigned to buttons that are easier to press Configuration Assign 3 Depending on the microscope configuration settings must be done manually if necessary l l l Fig 13 Configuration panel 04 2009 11 Configuring the beam path and lasers e Click the Acquisition button 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 Dye DAPI Alexa Fluor 488 td Tomato tdTomata Cya Alexa Fluor 483 Best signal none 1 ANS 2 Methylbenzoxazole 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 best compromise between both s
5. Series Bleaching on Speed Tile Scan A i Max gt a r T egions Pixel Dwell 6 40 psec octan Time 491 52 msec Online Acquisition Acquisition Mode how all 7 Avera g ing Channels Focus owal A Number Bit Depth o Bit Stage Multidimensional Acquisition i Information On Experiment Fig 20 Acquisition Mode tool Adjusting scan speed 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 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 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 04 2009 17 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 e Select the Channels tool in the Left Tool Area
6. 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 automatically in X and Y by first conducting a coarse and then a fine alignment Use the Models tool to define model equations to which measured data can be fitted You have three options to define a model 24 Correlation assemble a correlation model from predefined equations which will be fitted analytically PCH assemble a photon counting histogram model which will be fitted numerically Formula program a user defined model equation which can be fitted analytically 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 MBS or secondary dichroic beam splitter SBS 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 E System Configuration Showall ie Light Path Pinhole Pupil filling iO hE Diameter i 35 0 96 Airy Units Latest Adjustment Adjust Pinhole Pinhole Diameter pm Auto Adjust Position
7. 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 identify 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 measurement completion the data is displayed in the FCS Correlation diagram within an Image Container see Fig 35 04 2009 25 CC 486 633 x Fes Correlation Count rate kHz 30 40 s 60 Measurement Time s CyS RBG Crass correlation Chi vs Gh Correlation 4 5 Frequency 1400 1200 1000 B00 BOO 0 00010 00030 00100 003 0 010 0 03 Pulse distance ms 0 10 ys RAE ross correlation Chl vs Ch2 Frequency 1000 0 001 Lag Time 3 0 01 Counts binning tirme 3 RhGG 26 ms Cys RBG Cross correlation Chi vs Cha Cross correlatian Chl vs Cha Repetition Countrate Counts per Correlation Amplitude KHz Number particles Triplet state Triplet state Fraction 26 27 0635 Translation Structural pararr Component Component
8. 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 trom a number of options and proceed to the WINDOWS Save As window to save the exported data to disk 04 2009 Using the ConfoCor 3 module e Click on the Acquisition button e Use the ConfoCor Tool Group in the Left Tool Area to acquire and analyze FCS data Count rate z 5t n snap Start ConfoCor Experiment Fig 30 ConfoCor Tool Group E e Open the System Configu Light Path ration tool to define the light CC 488 633 ale ale path laser lines and pinhole 2 Channel Module position al The Light Path and Pinhole panels of the System c Configuration window display the selected track ae configuration which is used for the FCS procedure and the aa pinhole size see Fig 31 Invisible light ie vi ACCh2 EP 5406410 iR CCChI gt Ch CC Ch gt Ch t Laser Lines t Pinhole Fig 31 The ConfoCor 3 Measure Tool System Configuration 04 2009 23 You can change the settings of this panel using the following function elements Visible light Set the Pinhole diameter via slider or input box Press the Count rate button to open the Real Time
9. 1 Relaxation tire Fraction Diffusion time Hs Hs 111 702 490 318 molecule kHz 5 990 5 496 0 917 mS eno Walle See E 2 000 E 5 2 ULL EA ES ES EN EN Fig 35 FCS Correlation diagram You have the following function elements Fes Correlation Activate the FCS Correlation panel to display measured data Fig 35 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 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 Save Data F 26 04 2009 Reuse i Pressing the Reuse button will set the system configuration to exactly the same values t 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 The acquired FCS data is analyzed in the Fit display of the FCS diagram see figure Fig 36 CC 446633 x Correlation Model 3DireeiCiTnw 4 5 Channel RBG Parameter Number particles Am p litude i Geo metric correct
10. Fig 32 Pinhole adjustment T Models Model Correlation Offset Background Amplitude Antbunc hing Triplet Rotation Transtation Flow Stretrhed Exponential Git 1 Fig 33 Models tool 04 2009 Starting a measurement a Acquisition e Select the Acquisition toolbar t Times Calculated Durabon 1 min 40 s t Konetics t Positions The Times Kinetics and Position panels of the Fig 34 The ConfoCor 3 Measure Tool Acquisition toolbar display the selected Acquisition 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 Mes Count rate i P Start ConfoCor Experiment Enter the Bleach Time Measureme Time and Repeat Count into the corres ponding input boxes Activate deactivate a kinetic procedure by ticking the Use Kinetics check box Enter the time distance between measurements the cycle number and the shape in the corresponding input boxes Select the carrier and the sample or laser position by the stage or by the scanners 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
11. Microscopy from Carl Zeiss LSM 710 LSM 710 NLO and ConfoCor 3 LSM Software ZEN 2009 April 2009 We make it visible Page NS ta stro ete EEE E 1 BACCO anasto tapes inten neers nostic mesenteric eee deans A E sneete en 1 Starting the System casas ee ts ase te tes ne pss ame ee eames pea ee neces een ene 2 Introduction to ZEN Efficient Navigation ccccsccseesssseeeeeeeeeeseaneeesaeseeeseaseessoeneeesonaaes 5 Setting up the WCU OSC Cae e tertecetc cetera vacant ea atppc eve toned ene nctia sa cvacduniiceceeeeimucevandcueeeecie 10 Configuring the beam path and lasers cccecssceeeseeeeeeeeseeeseeeeessaeseeesaaeesseaseesseaseeessoes 12 Scanning an WNC CO ocecoc rece tire ocavecie notion sieceanecounecowetiocateacsmaocsrecemetdareiaandcuss ward etaueiaete end mca ecdeett 17 Storing and exporting image data cccceeeeceeseeceeeneeseeneeseeeeeseeneesaeeesaaeeseeneessoenessansessnaes 22 Using the ConfoCor 3 module cst ssettsctectccetec seer teeeiee tee rtect eet eee eee eee 23 Switching Bee Wik a ek C 9 eee eee eer eee ee eee eee eee ee eee 29 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 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 sample
12. an 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 04 2009 5 Workspace zoom Variable number of columns f Light Path Channel Mode Undock tool Fig 7 ZEN Window Layout configuration More features of ZEN 2009 include The user can add more columns for tools to the Left Tool Area or detach individual tools to position them anywhere on the monitor To add a column drag a tool group by the title bar e g Online Acquisition to the right and a new tool column automatically opens Alternatively use the context menu move toolgroup to next column To detach a tool click on the little icon on the very right end of the blue tool header bar Fig 7 Another unique feature in Imaging Software is the scalable ZEN interface This Workspace Zoom allows adjustment of the ZEN 2009 window size and fonts to the situational needs or your personal preferences Fig 7 Setting up conventional confocal software for a specific experiment can take a long time and is often tedious to repeat With ZEN these adjustments have to be done only once and may be restored with just two clicks of the mouse For each type of experiment one can now set up and save the suitable Workspace Layout These configurations can also be shared between users For most controls buttons and sliders a tool tip is available When
13. d 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 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 04 2009
14. 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 i Tracks Channels TY Taki Select all Unselect all Tracki Wf vw 405 488 505 561 633 405 nm y 6 5 Continuous Wave Attenuation OFF TD Acquisition 1 0 Configuration A Zeiss LSM 32 0 Smart Setup Show manual tools E Be Lo i Auto Exposure Continuous Snap Digital Offset Z Stack Time Series Ligita sain ah dk 4k Bleaching Tile Scan 80 00 KB Gain Ma Regions Digital Offset 4k 4k 4k Online Acquisition A b Digital Offset as dE dbj dH Multidimensional Acquisition gt L Fig 21 Channels tool 18 04 2009 Image acquisition Once you have set up your parameter as defined in the above section you can acquire a frame image of your specimen e Use one of the Auto Exposure Live Continuous or Snap buttons to start the scanning procedure to acquire an image e Scanned images are shown in separate windows e Click on the Stop button to stop the current scan procedure if necessary a Select Auto Exposure for automatic pre adjustment of detector gain and offset Auto Exposure Select Live for continuo
15. flow 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 Time Series pag Show all mode active owal 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 A Cycles Interval Trigger In Trigger Out None v None v Fig 6 Show all 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 th
16. g Cc 0 75 MB Rabb aortic SMC jpg C Channel Mode 0 75 MB lt lt rokortexcalesurn jpg Trocki C x 4 0 87 MB sen cplaques jpg Cc 0 57 MB shrimpbinstuls jpg 700 gt C X 7 co Gomes PMT 1 None PMT 2 None New image cpesrectonC anges po i w ve wi v 1 1 MB Dimensions E now af Standard ITCBOOIPYCy2 sm Zoom D 0 25 MB StandardRhodaminCy3ete Ism 0 25 MB Fig 8 New image document in the Open Images Areas 04 2009 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 file 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 Desktop Axiovision 4 6 Icons demo data 4D cells fromChris Nice Small LSM Demo Image Gif Movies aoa Riken Meta images Kogure Best mdb vonZIKUP ZIKUP_ImageData mdb JenaKultur JofBioTech_CallforPapers LSM 4 2 Icons Movies Music 0 60 MB narration zen temp install ZEN2007 AIM AIM45 Daten_Olaf Documents and Settings Administrator All Users Default User HPPET M10SE Application Data Desktop Axiovision 4 6 Icons amp demo data 4D cells fromChris cervicalsmear amp Nice Small LSM D MB Gif Movies 0 58 MB File Browser 3 Channels 68 bit 3 Cha
17. igital 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 04 2009 Scanning a Z stack A continuous XY scan of the set focus position will be performed 7 Chon be 7 Stack V Show all 7 Select Z Stack MESSA in the main tools area First Last Center Open the Z Stack tool in the Left Tool Area Set Last Select Mode First Last on the top of the Z Stack tool 0 00 um Click on the eee button in the Action Interval Slice Button area i Set First 10 00 0 00 gt Optimize Sectioning and Step Use the focus drive of the microscope to focus on the upper position of the specimen area where the Z Stack is to start gt Correction Click on the Set First button to set the upper Fig 26 Z Stack tool 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 Start Experiment Click on the Start Experiment button to start the recording of the Z Stack K 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 i
18. io n TE state Frai ction 3 Relax ation time Hs 4 119 vl omponent 7 ee os Fraction Diffusion time us 0 001 0 01 1k Structural pa rameter ee Time s Fitrange 1 40 sto 3 36 5 Fit Fit All Undo Reda To Method Repetition Countrate Counts per Correlation Amplitude Triplet state Triplet state componenti Component1 Translation kHz molecule Number particles Fraction Relaxation time Fraction Difusion time Structural pararr kHz us 3 us 5 496 5 990 2 090 0 917 27 063 4 062 111 702 5 000 1 908 0 508 190 318 5 000 arene 0 731 27 999 359 169 416 5 000 meege 81728887 2o ossz aisee Anis oesoo gage S00 1 794 0 558 178 465 5 000 EA ES ES ES ES Fit Diagrama ER Fit diagram Fit deviation diagram Fig 36 FCS Fit diagram 04 2009 27 You have the following options Activate the FCS Fit panel to display fitted data Fig 36 Model model name Parameter 28 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 define
19. nnels 8 Arabidopsis 3 Channels 8 bi 75 kB 65 kB 0 57 MB ciliate 3 Channels 65 bit B Alzheimer RETF LIM 3 Channels 6 0 15 MB 0 57 MB 3 Channels 8 bi 0 vB 87 MB candidaalbicans 3 Channels 68 bit 0 17 MB 0 75 MB icmousekidney cysticproximaltubules 3 Channels 8 bit 3 Channels 8 bit 0 11 MB 0 75 MB 0 75 MB AlzheimerFRETFLIMs 3 Channels bit 24 kB 1 1 MB centrin 3 Channels 85 bit B 0 56 MB Daphn 3 Channels 8 bit alzheimers 3 Channels bit 0 30 MB 1 1MB ra cerebralarmyloidangio 3 Channels 8 bit I uB 0 87 MB DAPI 3 Channels 83 bit 48 kB 0 44 MB 04 2009 Turning on the lasers ZEN 2009 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 manual tools tickbox and open the Laser tool All available lasers can be operated within this tool Fig 10 Laser GRRE las Acquisition Argon Configuration DAPI DPSS 561 10 HeNe5do4 Smart Setup Show manual tools Le co 6 ew Auto Exposure Live Continuous Snap HeNebs3 Diode 405 30 Z Stack Time Series Bleaching Tile Scan Positions Regions Laser Properties Online Acquisition y Acquisiti
20. on Mode Maximum Power Channels Focus Wavelength Status Multidimensional Acquisition i Information On Experiment gt Fig 10 Laser Control tool 04 2009 690 1024 456 488 514 561 594 633 F 50 mW 633 nm connected Setting up the microscope Changing between direct observation and laser scanning mode The Ocular and Acquisition buttons switch between the use of the LSM and the microscope Fluorescente Ay Acquisition Configuration A Zeiss LSM Smart Setup Show manual tools a Ga a Auto Exposure Live Continuous Ocular EC Plan Neofiuar 1000 3 Aperture 0 16 HF e Click on the Ocular button to 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 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 e Open the graphical pop up menu by clicking on the Objective symbol and select the objective lens for y
21. or 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 A A click on this arrow button will move the selected track highlighted in light grey one position downwards in the list box 16 04 2009 Scanning an image Setting the parameters for scanning 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 The number of pixels influences the image resolution 73 Acquisition Mode x Prete aea Objective EC Plan Neofluar 10x 0 3 Configuration A Zeiss LSM ocan Mode Frame Smart Setup Show manual tools Frame Size x 128 e New Auto Exposure O ti pima Z Stack Time
22. our experiment Fig 11 e The chosen objective lens will automatically move 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 Fig 11 Microscope Control window e g e Click on the On button Set the intensity of the Axio Imager Z2 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 04 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 ee Se Fherescence SRHEFOR Shutter or 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 Transmitted light control e Click on the Reflector button and select the desired filter set by clicking on it Storing the microscope settings Reflector Microscope settings can be stored as configurations Fig 13 by typing a config name in Reflected light the pull down selector and pressing the save as snnier EO Pian Neofuar button Fast restoration of a saved config is achieved by selecting the config from the pull
23. peed 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 12 04 2009 Best signal Best compromise Linear unimixing Emission signal Speed Pye r n PN Em a l 10 Cancel Fig 15 Proposals panel of the Smart Setup tool Pressing Apply automatically sets the hardware parameters in the displayed way for the dyes chosen If the option Linear Unmixing is selected the system is set in the lambda mode automatically Pressing the Auto Exposure Wmekmusisi button will then optimize the settings of the Gain Master and offset for the given laser power and pinhole size Further image optimisation from this point can be done easily 04 2009 13 Setting up a configuration manually 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 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
24. s 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 04 2009 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 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 Power remote switch e Switch on the main switch of the X Cite 120
25. s activated the Start Experiment button is grayed out and only single images can be scanned 04 2009 21 Storing and exporting image data Fig 27 Save Image buttons in ZEN speichern unter Speichern E Desktop o w Eigene Dateien demo data Y Arbeitsplatz 9 Jenakultur e Metzwerkumgebung C JofBioTech_CallforPapers 9 CiscoSecure Authentication Agent 35M 4 2 Icons a Divs Movies 9 Movies fom 4xiovision 4 6 Icons fm Music lgl gt Dateiname Image Z Dateityp o Abbrechen Fig 28 Save as window Tagged Image File X Contents of image window single plane Raw data series Contents of image window single plane Contents of image window series Full resolution image window single plane Full resolution image window series Select file name and save Fig 29 Export window 22 e To save your acquired or processed images click on the Save or Save As button in File Menu or click the a button in the Main Toolbar Fig 27 1 or click on the EJ 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 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
26. shown in Fig 16 E Light Path s Showall 4 Channel Mode Lambda Mode Online Fingerprinting Tracki Acquisition Configuration DAPI Smart Setu ae p Seil eenen ths Color Detector Range A x Chi 408 507 nm New Auto Exposure i Continuous Snap ys Chs1 35 691 nm Che 415 735 nm Reflection Z Stack Time Series Bleaching lt a Tile Scan Plate ae Positions Visible light oe EMBS 4552k Invisible light Online Acquisition Acquisition Mode eae A Channels t Focus lt gt Stage Stage Focus Multidimensional Acquisition s t Ratio i Information On Experiment Fig 16 Light Path tool for a single track LSM 14 04 2009 e Select Channel Mode if necessary Fig 17 The Light Path tool displays the selected track configuration which is used for the scan procedure e You can change the settings of this panel using the following function elements assigning a color to the channel Select the appropriate filters and activate the channels Click the Laser icon to select the laser lines and set the attenuation values transmission in in the displayed window For the configuration of the beam path please refer to the application specitic configurations depending on the used dyes and markers and the existing instrument configuration In the Imaging Setup tool the Detection Bands amp Laser Lines are displayed in a spectral panel Fig 18 to visualize the acti
27. tart System Offine Demo Cancel c LSM 710 Startup window Fig 3 ZEN 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 Start System hardware for acquiring new images or in Image Processing mode to edit already existing images Toggle the little o symbol to view the Boot Status display and get the additional Offline Demo button option Afte Choosing Start System initializes the whole microscope system and activates the entire software package for new image acquisition and analysis The Image Processing mode ignores all hardware and activates only data handling and image processing functionality for already acquired images The Offline Demo mode reads the current hardware database but does not activate the system hardware for use Instead it simulates the system hardware for training purposes Upon clicking the Start System button the Image Processing 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 04 2009 3 A Application Bar B Menu Bar C Main Toolbar D Left Tool Area E Center Screen Area hosts up to 3 3 Containers F Right Tool Area G Status Area Fig 4 ZEN Main Application Window after Startup with empty image container
28. 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 for the ZEN 2009 software please refer to the User Manual that is provided with your system 04 2009 To create a new image document in an empty image container click the Snap MES or the Auto Exposure button For an empty image document press the New r button The new document is immediately presented in the Open Images Area Remember an unsaved 2D image in the active image tab will be over written by a new scan Multi dimensional scans or saved images will never be over written and a new scan will then automatically create a new image document Acquired data is not automatically saved to disc Make sure you save your data appropriately and back it up regularly The ZEN software will ask you if you want to save your unsaved images when you try to close the application with unsaved images still open 23 There is no image database any more like in the earlier Zeiss LSM software versions a ZEN 2009 Fie Acquisition Maintain to s V Workspace Zoom th B iy Workspace Configuration Configuration DAPI i poamecimi ipg SmartSetup J Show manual tools c 0 58 MB e Auto Exposure polytenechromosomes jpg Cc 0 58 MB Proliferatinghe mangioma jpg C 0 52 MB Rabbtaoric SMC 1 jp
29. us 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 Fig 22 Image Display 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 23 23 Clicking on the right hand side of the button leads to a list of colors 04 2009 View Dimensions Z Position 1 Zoom Channel s Merged Chi Fig 23 View Dimensions Control Block 19 Fig 24 Image Display Channels v Showa 4 Wf Tracki Unselect all Tracki vi 405 65 Continuous Wawe Attenuation OFF 55 70 561 nm f 10 32 0 1AU max Fig 25 Channels tool 20 The scanned image appears in a false color presentation Fig 24 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 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 D
30. vated laser lines for excitation vertical lines and activated detection channels colored horizontal bars 04 2009 Settings for track configuration in Channel Mode fA Imaging Setup Showall A Mode Channel Mode r simultaneous Switch track every Frame W Tracki Track not defined Fig 17 Imaging Setup tool for a single track LSM Activation deactivation of the excitation wavelengths check box and setting of excitation intensities slider If necessary open the Laser Control tool see above Selection of the main dichroic beam splitter 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 H Imaging Setup V Showall 4 Mode Channel Mode simultaneous Switch track every Frame V AF488 TdTo A488 tdTo AF488 TdTo Detection Bands amp Laser Lines display Fig 18 Configuration CH1488 e For storing a new configuration click and enter a desired name in the first line of the list box Fig 19 then click Ok to store the con figuration Save cument acqueition 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 ees Fig 19 Track
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