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CHAPTER 5 - MULTIPHOTON LASER SCANNING …

LSM 880

Non Descanned Detection (NDD)

ZEISS

10/2014 V_01

000000-2071-464

In addition the power connections have to be made in the following way to the plugs on the back of the

electronics rack:

Fig. 9

Power connections of NDD detectors and the scan modules on the electronics rack of
the LSM NLO.

Summary of Contents for LSM 880

Page 1: ...LSM 880 LSM 880 NLO Operating Manual October 2014 ZEN 2 black edition...

Page 2: ...specifications are subject to change the manual is not covered by an update service Unless expressly authorized forwarding and duplication of this document and the utilization and communication of its...

Page 3: ...which will help you to find out which settings and methods are capable of improving or accelerating a procedure The HOT SURFACE symbol warns against hazards for the user that might arise when touching...

Page 4: ...s e g maintenance macros 3 Additional Software Tools This section contains a description of the use of additional tools for setting the microscope 4 Annex The annex contains the Application specific C...

Page 5: ...t wavelength for bleaching might result in the first image s following the bleach event not being final as the GDD can still be moving This can be set via database Contact your ZEISS representative on...

Page 6: ...h step sizes of 10 nm lead to faulty processing Choose 100 nm steps or above as suggested by software for Z Stack acquisition When analyzing PALM data for particle tracking it is possible to select an...

Page 7: ...Performance Features of the LSM 880 14 2 6 Microscope Equipment of the LSM 880 15 2 7 Computer Hardware and Software 17 3 STARTUP AND SHUTDOWN OF THE SYSTEM 18 3 1 Startup of the System 18 3 1 1 The P...

Page 8: ...nsional Acquisition Settings 75 5 2 5 Setup Manager Laser 77 5 2 6 Setup Manager Experiment Designer 80 5 2 7 Setup Manager Imaging Setup 84 5 2 7 1 Imaging Mode and Track Definition 84 5 2 7 2 Filter...

Page 9: ...Scan 152 5 2 18 Scan Overview Image for Tiling 154 5 2 19 Multidimensional Acquisition Time Series 155 5 2 19 1 Interval Time 155 5 2 19 2 Marker 157 5 2 19 3 Start 159 5 2 19 4 End 161 5 2 20 Multid...

Page 10: ...3 19 Snap 259 5 3 20 Start Experiment 259 5 3 21 Taking FCS Measurements 260 5 3 21 1 Start and End of a Measurement 263 5 3 21 2 Loading Stored Data Files 267 5 3 21 3 FCS Correlation Diagram 268 5 3...

Page 11: ...19 3 Online Multiview Processing 381 5 4 20 Processing Copy 382 5 4 21 Processing ICS 384 5 4 21 1 Detrend 384 5 4 21 2 Remove Structure 385 5 4 21 3 ICS Correlation 386 5 4 21 4 Map Filter 387 5 4 21...

Page 12: ...sions 424 6 2 2 Display 427 6 2 3 Player 428 6 2 4 Graphics 429 6 2 5 Preview View 432 6 3 Split View 433 6 4 Gallery View 436 6 5 Airyscan View 437 6 5 1 Airyscan View Option Tab Dimensions 437 6 5 2...

Page 13: ...leaching 503 6 18 2 Tools in the FRET View Options Control Block for Sensitized Emission 505 6 19 Raster Scanning Image Correlation Spectroscopy RICS 508 6 19 1 Access to RICS 509 6 19 2 Control of Vi...

Page 14: ...s display edit and analyze the images It is a special user interface desktop based on the network capable graphic 64 bit Microsoft WINDOWS 7 operating system Portions Copyright 2007 Microsoft Corporat...

Page 15: ...iff LSM Tiff BMP Palette files ZEN Palette lut The backup of further hardware calibration settings including the database and licence file can easily be created with the CanCheck tool C ZEN CanCheck C...

Page 16: ...focus inside the specimen and the pinhole are situated at optically conjugate points Confocal imaging The decisive advantage of this arrangement is the fact that essentially no other light than that c...

Page 17: ...time gives the residence time of the molecule in the confocal volume and hence its diffusion time If the two interacting molecules are of different size only the smaller one has to be labeled using fl...

Page 18: ...her degree The resolution reaches 140 nm in x and y and 400 nm along the optical axis The emission signal is filtered using standard emission filters The detected signal is processed online for immedi...

Page 19: ...which separates the emissions from the excitation light In the LSM 880 the fluorescence is either spectrally separated by passing through a grating onto the Quasar detector 3 PMTs or 2 PMTs plus 1 ce...

Page 20: ...registration of multiple fluorescence signals identical optical sections can be obtained in each confocal channel The microscope s transmitted light channel is equipped with a photomultiplier too It i...

Page 21: ...f you want to use more than five conventional fluorescence filter sets it is advisable to use a further reflector turret When changing the reflector turret position make sure that the turret will clic...

Page 22: ...pport Alternatively the light is directed to the LSM transmitted light detector or enables conventional transmitted light observation The focusing screen for conventional transmitted light is located...

Page 23: ...s once set or complex examination sequences once established can be saved and reproduced therefore complete application programs can be loaded and performed by pushbutton control The software of the L...

Page 24: ...erating manual and CHAPTER 4 ANNEX for handling of any Titanium Sapphire Laser used for Multiphoton NLO Microscopy 3 1 1 The Power Remote Switch To start the system switch the main switch Fig 5 1 to O...

Page 25: ...systems The optical beam path relative pinhole position and scanner adjustment can be set and checked automatically on the systems ZEN Configuration Tool icon The Configuration Tool permits a new or...

Page 26: ...t Once the program is loaded the Login ZEN dialog will appear Fig 7 in front of the ZEN interface The number after ZEN designates the current version By pressing the question mark symbol the context s...

Page 27: ...boot process will be displayed by the blue time tab The single components will be listed which are checked during the boot process Note that the software will not control the hardware in this mode Ima...

Page 28: ...oose button Windows Explorer will open Search for the folder file the database is stored and and open it Select it for use Select a database suited for your purpose from the Explorer window You can al...

Page 29: ...oftware is still active or else you will lose the currently set operating parameters and the images just scanned In the Maintain tab System Options dialog window activate Laser off on Exit in the Shut...

Page 30: ...the lasers will be automatically switched off when exiting the ZEN program If the box is not ticked the Laser Off dialog box will appear that allows one to check on the listed lasers Fig 14 By the Pow...

Page 31: ...ZEN software window This terminates the ZEN software Shut down WINDOWS About 20 seconds after WINDOWS is shut down your computer turns off Please bear in mind that a cooling phase of at least 5 minute...

Page 32: ...ut LSM 880 26 000000 2071 464 10 2014 V_01 4 Introduction to the Software Application Layout 4 1 Overview on the Screen Layout Fig 15 ZEN Main Application window after Startup with empty image contain...

Page 33: ...low 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 vie...

Page 34: ...oncept ensures that tool panels are never more complex than needed In the basic mode of the tools Show all is deactivated and the tools show only the most relevant functions covering approximately 80...

Page 35: ...ar to where you want it Fig 18 Another feature is the scalable ZEN interface This Workspace Zoom allows adjustment of the ZEN window size and fonts to the situational needs or your personal preference...

Page 36: ...on Tool group Tool closed or tool window opened Panel e g Speed panel Field with a subset of tools of a tool window List box or selection box Selection of one of the displayed options via a mouse clic...

Page 37: ...moving via the arrow keys of the keyboard Press the Shift or Ctrl key while clicking on the arrow button to change the numeric values in coarse or fine steps Button Selection performance of a functio...

Page 38: ...ins the menu items and the controls for the Workspace Zoom including the Reset button for the Workspace Zoom and the button to re dock all free floating tools to the Left Tool Area Fig 19 Menu bar lef...

Page 39: ...ition Document Opens a new and empty image document Open Opens an existing document in the active image container Save Saves the active document Save As Saves the selected image with entering new form...

Page 40: ...ve container see section ZEN File Browser In the second group of entries in the list the five last accessed files are listed Selecting a list entry will open the respective file In the third group of...

Page 41: ...rtite On upper section lists user accessible tools the lower section lists tools reserved for service personnel they are password protected See also section Maintain Tab for further information 4 5 2...

Page 42: ...the mouse cursor in the camera image The Set button will become active Press set to store new settings press Reset to return to the old settings Use the arrow buttons to adjust the color balance of t...

Page 43: ...V_01 000000 2071 464 37 4 5 3 Help About The About window can be accessed via the Help item in the menu bar clicking About opens the following dialog Fig 25 About window The panel hosts important inf...

Page 44: ...ng saving or deleting a workspace configuration These workspace configurations are saved settings that allow to restore a pre defined layout of the whole ZEN application window including status size a...

Page 45: ...01 000000 2071 464 39 To load a workspace configuration click on the button The following list box will appear Selecting on of the list entries will load the respective configuration To delete a works...

Page 46: ...tails of this one are shown If more than one process is running then the single process information will be shown in the gallery Fig 29 Progress bar System information Shows information of the current...

Page 47: ...ion Parameter Setup Manager The tools of this tool group Laser and Setup Imager are only visible if the check box Show all Tools is checked Those tools are necessary if the system should be operated i...

Page 48: ...multidimensional acquisition tool group The tool is then active and will be used after hitting the Start Experiment button The experiment type is represented above the button by a graphic Tools tools...

Page 49: ...back with one click use the button in the top right corner of the menu bar To position the tool window freely on the desktop use the drag and drop function Open a tool window Clicking on the tool head...

Page 50: ...e procedure Live starts the fast scanning procedure scans with lower resolution and higher speed Continuous scans an image continuously with the currently active scanning parameters Snap scans one sin...

Page 51: ...available image views Using the context menu of the Center Screen Area by clicking the background of a container the view of this area can be varied individually The Center Screen Area can be split in...

Page 52: ...image view field of all containers in vertical direction for a larger image display Clicking the arrow up button shows the image area in all containers once again Image view controls Activates one or...

Page 53: ...ns the context menu of the Center Screen Area The following functions are available Display of the image tabs in the container header Three options are available Text View Small Thumbnail View Large T...

Page 54: ...decreasing the center tool area Dragging the slider to the right side decreases the Right Tool Area up to the default size Save button Saves the selected scanned or changed image The Save as dialog ap...

Page 55: ...ation Layout ZEISS 10 2014 V_01 000000 2071 464 49 Big View button Shows images with the textual characters and a big image preview Save Status icon This icon appears in the image that is not saved ye...

Page 56: ...tabs Fig 40 Locate Visual and camera inspection in transmitted and epifluorescence light Acquisition Imaging with LSM or ELYRA FCS Fluorescence Correlation Spectroscopy LSM 880 only Processing Process...

Page 57: ...configuration from the Configuration selection box by pressing the Load button All saved ocular configurations will be listed in the Load Configuration panel Fig 42 Select the appropriate one by highl...

Page 58: ...ailable The configuration that should be assigned to a button can be selected from the Configuration selection drop down menu By pressing Assign the configuration will be assigned to the button that w...

Page 59: ...ation you have to expand the Microscope Control tool by pressing the expansion symbol in the Microscope Control tool tab Fig 47 The Microscope Control panel contains the following functions HAL On Off...

Page 60: ...position of the filter can be selected from the Filter drop down menu only for motorized condensers Stage button If the Stage button is pressed the Stage control panel will appear Fig 51 By pressing...

Page 61: ...er the nose piece z position for objective change Work brings the z position back to the actual used position if the focus was zeroed or in Load position Home and Load positions can also be accessed f...

Page 62: ...aried via the input box or slider The light path will be highlighted by color in the diagram Press the On button again to shut down the light The button will go to the Off position and the shutter clo...

Page 63: ...ate tab of the main tool bar is pressed When working with Airyscan we also recommend using selected high NA objectives For reflected light observation Epi fluorescence Turn on the Excite power supply...

Page 64: ...e required specimen plane Select specimen detail by moving the stage in X and Y via the XY stage fine motion control Fig 59 3 and 2 2 Microscope settings on Axio Observer for transmitted light observa...

Page 65: ...upply switch Fig 59 1 Click on the Reflected Light button and set the shutter to Open Click on the Reflector button and select the desired filter set by clicking on it The filter is automatically move...

Page 66: ...atically set when switching between Locate and Acquisition in the Main toolbar Reflector Push and click reflector cube can be selected via graphical pop up menu Objective Objectives can be selected vi...

Page 67: ...ightness of the halogen lamp with the potentiometer Fig 61 4 or the Intensity slider in the Microscope Control tool Set the required transmission value of the gray filters in the Filter frame Set the...

Page 68: ...he required objective as follows Open the graphical pop up menu by clicking on the Objective button Click on the objective you want to select The selected objective will automatically move into the be...

Page 69: ...Click on the desired reflector module The reflector turret moves the selected reflector module into the beam path The FITC filter set consists of an excitation filter for the 450 490 nm spectral range...

Page 70: ...ture will also be displayed The Atmosphere panel allows defining the O2 and CO2 concentration via the input boxes either type in the numbers or use the arrows to set the parameters In order to monitor...

Page 71: ...ning microscopy LSM It depends on the hardware configuration which of these functions are available All wide field techniques use cameras whereas LSM uses internal photomultiplier tubes PMT or GaAsP f...

Page 72: ...ng it in the Recent section which lists the recently used acquisition parameters or from the Parameters section where all saved acquisition parameters are displayed To search for an acquisition parame...

Page 73: ...sition parameters in the data base or press Cancel to abort the process Pressing the Smart Setup button will open the Smart Setup panel As this is an exclusive LSM function the user is referred to sec...

Page 74: ...Setup Fig 70 provides an intuitive user friendly interface for automatically configuring the LSM s light path for detecting fluorescent dyes Similar to cameras used in photography a panel of motifs he...

Page 75: ...dyes is done by typing the name of a dye into the Search box and then selecting it from the list Alternatively the most recently used dyes can be picked from the upper section of the panel A color re...

Page 76: ...In the Acquisition tool the image size shows close to niquist settings in xy a slow scan speed 12 bit data depth and unidirectional scan mode without averaging Clicking apply sets the parameters and s...

Page 77: ...een the acquisition tracks This proposal favors speed over cross talk The proposal for Best Signal follows a strategy that maximizes the efficiency of signal recording and also minimizes the level of...

Page 78: ...plays a bar chart representation of emission signal cross talk and speed with each proposal The color coding specified for the stains is used for displaying the bar charts Fig 74 Smart Setup displays...

Page 79: ...tton active Set Exposure optimizes the gain and offset settings PMT only for cameras only the exposure time will be adjusted of all active channels and tracks dependent on the given laser power Set Ex...

Page 80: ...es after its activation Fig 80 Fig 80 Action buttons Continuous button active Snap produces one single image using all active channels and tracks thereby ignoring the parameters activated in the multi...

Page 81: ...ield of views FOV Positions Acquisition of an image series at different positions Regions Imaging or manipulation of defined regions of interest ROI The settings for multidimensional acquisition are a...

Page 82: ...latter can be however manually deselected if required Pressing the Start Experiment button will trigger the image acquisition taking into account all activated functions The progress of the experiment...

Page 83: ...ll be switched on automatically after software start The Laser Properties panel displays the current wavelength status mode ps or cw if applicable tube current Argon laser and power Argon and Ti Sa la...

Page 84: ...nd warm up and being air cooled needs also some minutes to cool down after it has been switched off The lifetime is dependent on the the operation mode and messages are provided to help you run it in...

Page 85: ...still needed for the warm up of the laser until it is either On or in Standby During this time and while in Standby the laser wavelengths are marked in red in the Channels tool window to indicate that...

Page 86: ...s achieved via AOTF The amount of power is proporational to the electric power generating the acousto optic wave in the AOTF cristal The AOFT cristal can deal with up to 8 laser lines in parallel Howe...

Page 87: ...ion block is present it cannot be disabled Disabled blocks have to be enabled first before they can be activated New Blocks are added using the Create drop down list A Acquisition Block is used for im...

Page 88: ...ment Blocks dialogue chose a name from the Experiments list with mouse click Select All or Deselect All blocks using the corresponding buttons Single blocks are selected deselected with a mouse click...

Page 89: ...k as the reference position For a given Z Stack go to the center position and if necessary click Center within the Center tab of the Z Stack tool once to confirm the reference All imaging modes define...

Page 90: ...nd saved for the different imaging modes Click on the Imaging Setup tool or close the tool window Fig 95 The Imaging Setuptool is divided in the Imaging mode selection Beam path configuration Ratio Im...

Page 91: ...deactivated this track will be deactivated too With the Switch track every drop down menu only available if Show all is active you can select the way tracks are switched The different tracks can be sw...

Page 92: ...ded for the LSM imaging mode and must be assigned to position 1 as this position wil be automatically selected in the LSM imaging mode The other position position 2 can be equipped with a cube that is...

Page 93: ...Position m and Y Position m display boxes show the actual x and y coordinates You can edit the numbers by typing or using the arrowheads If numbers were altered the first click on the compass rose wil...

Page 94: ...e H or L buttons The z position can be altered by grapping and sliding the plane or by using the arrow slider The plane shows the actual z position Pressing Manually in the Zero position field will se...

Page 95: ...X Cite or HXP lamp can be varied via the input box or slider The light path will be highlighted by color in the diagram Press the On button again to shut down the light 5 2 7 8 Aperture Control When...

Page 96: ...lation are set in the Channels tool of the Online Acquisition tool group A suitable color can be assigned to each of the two Ratio Channels R1 and R2 if their icons are pressed Fig 108 The scroll bar...

Page 97: ...e imaging set up Activate the detector channels by checking the box es in the Use column Detectors can be added removed depending on the hardware clicking the plus minus button Assigne a Dye to the de...

Page 98: ...on spectrum for each pixel corresponding to the emission spectrum of a specific dye This calculation is done by linear unmixing see section Unmixing View using the acquired Lambda stack and allows to...

Page 99: ...ct it from the drop down menu Reflector Turret The filter wheel FW1 is the reflector turret of the microscope that holds push and click filter cubes and fixed beam combiner cubes When using the LSM fo...

Page 100: ...changed as required It is also possible to load already stored track configurations single tracks in a recording configuration Available in the Channels tool window The setup is done using both the Ch...

Page 101: ...rom the drop down menu next to Source 1 and Source 2 As an alternative to a second channel for the ratio imaging is also possible to choose the first images of a time series of one channel for the rat...

Page 102: ...de scanning procedure with regard to the main dichroic beam splitter and the QUASAR detector settings are performed in the Imaging Setup tool Fig 115 The QUASAR detector is displayed for spectral imag...

Page 103: ...pectra together with the excitation settings allowing an immediate display of the unmixed images while scanning Chose Online Fingerprinting in the Imaging Setup tool You can change the settings using...

Page 104: ...ser lens aperture and filters and the tube lens The use of this function permits the use of a Zeiss AxioCam camera various models see ANNEX for description as an alternative external detector The came...

Page 105: ...checkbox in the beam path Fig 118 Make sure to set the filter wheel for internal and external detectors to a plate position or a dichroic which transmits the required wavelengths Mirror Emission Filte...

Page 106: ...individual settings can be configured for the Airyscan or the internal detectors In case Airyscan is used together with internal detectors only the switch track every frame mode can be used Laser and...

Page 107: ...e Imaging Setup tool Controls include Objective selection Scan Mode Frame Size and Line Step selection Format selection Imaging speed selection Averaging and color depth selection High Dynamic Range S...

Page 108: ...ize button available predefined n n or n m formats can be loaded directly Fig 125 It will depend on the detector type and camera chip which formats are available 5 2 8 3 Format Selection Binning of th...

Page 109: ...eraging n 0 4 or Continuous averaging The way of averaging can be selected in the Averaging Method drop down menu available only in Show all mode where you can either select Mean for the mean average...

Page 110: ...g for details on the properties and processing options of HDR files The HDR function is located under the field Averaging in the Show all mode of the Acquisition Mode tool Fig 130 HDR is activated thr...

Page 111: ...ction 5 4 12 HDR imaging does not produce usable results with high speed imaging 5 2 8 6 Scan Area Definition The Scan area when expanded displays the Image size and Pixel Size in m of the displayed f...

Page 112: ...s with different Z values Frame Z Stack Frame Z Stack Time Series scanning of XY frames in defined ROIs Frame ROI Time Series scanning of XY frames with different Z values in defined ROIs Frame Z Stac...

Page 113: ...the drop down menu clicking the button or type in the required values next to X and Y Recommended setting to start with 512 x 512 pixels It is also possible to enter different values for X and Y The...

Page 114: ...ans when moving backwards i e the Scan Time is halved The online scanner calibration OSC will correct for nonlinear distortions of the lines in bidirectional mode This function see also checkbox in Ma...

Page 115: ...range and immediately shows the changes made to zoom rotation and offset Set the desired Zoom factor via the slider or by clicking on the small red squares outlining the inner square The Zoom factor...

Page 116: ...ut delay of the image display Select 8 Bit or 12 Bit Data Depth i e 256 or 4096 gray values In the scan area it is possible to shift a sub region in the frame using the offset sliders if the Frame Siz...

Page 117: ...aw a straight line in the image at the position where the line scan should be performed Alternatively the scan line can be defined as a free shape curve spline The arrow selection is used to move the...

Page 118: ...e In the Spot mode fluorescent or reflected light occurring from a single voxel xyz is detected In this mode a spot can be defined by two perpendicular lines in the Image using the Spot Select button...

Page 119: ...100x magnification For optimal resolution the scan area should be centered and the zoom should have a factor of at least 1 7x or higher Zoom factors with negative influence on the resolution will be d...

Page 120: ...Acquisition mode dialogue You can either type in the required bandpass emission filters or select them from the drop down list Then press the OK button Fig 143 As a result a linewise multitracking wil...

Page 121: ...r Channels In the Channels tool window the following options are available Switching activating deactivating and naming saving of tracks Defining Detector settings Defining pinhole settings Defining D...

Page 122: ...activated deactivated for imaging with the check box Changes in the selection of the tracks in the Channels tool will automatically update the selection in the Imaging Setup tool and vice versa E g if...

Page 123: ...it will move one position down The arrows are only active if an upward or download movement is possible The sequence is updated in the Imaging Setup tool accordingly You can add up to 4 tracks by pres...

Page 124: ...track configurations are displayed To search for a track configuration type in its name or part of its name in the Search field and only those track configurations will be listed that contain the typ...

Page 125: ...gnation box automatically Track designation in the Imaging Setup tool will automatically be updated Fig 156 The total set of parameters for one or multiple tracks can be stored as an acquisition confi...

Page 126: ...ratio formation during the scan procedure If the input box does not contain any value at all or no suitable value the useful value last used will be activated The ratio channels are displayed as addit...

Page 127: ...value is defined by CRmax digital gain CRmax maximal count rate defined by the software which is 4 MHz for the BiG 2 and Ch2 and 2 MHz for CHs 32 channel GaAsP Array Higher count rates are regarded as...

Page 128: ...time 1 52 s 8 bit image Example With the GaAsP detector Big 2 software limits the maximum count rate to 4 Mhz 4 photons s At scanning speed 8 with a pixel dwelling time of 1 52 s one can obtain a max...

Page 129: ...in photon counting image Settings were digital gain 1 scanning speed 8 corresponding to a pixel period or pixel dwell time 1 52 s 8 bit image In the Status Area the displayed value of the pixel to wh...

Page 130: ...the Airyscan modes Detection gain is set in the same way as the internal detectors by the Gain and digital gain sliders SR VP CO Pinhole To set the system for increased resolution mode press the SR b...

Page 131: ...detection mode the required oversampling value for the pixel number and size changes This is adjusted automatically by using the Optimal button in the Acquisition Mode dialog Fig 167 after choosing a...

Page 132: ...Leveling in the Piezo field will center the Piezo stage position in regard to the microscope focus drive The Piezo Position m input box allows the position of the Piezo to be altered The Step Size m...

Page 133: ...ion and Autofocus Map cannot be used if an acquisition strategy includes tracks with camera based image recording The Focus Devices and Strategy tool Fig 169 provides controls for maintaining focus st...

Page 134: ...ass surfaces through laser reflection is only possible in the presence of a refractive index mismatch Readily detectable laser reflection occurs at the interfaces of either glass and air or glass and...

Page 135: ...s This information is stored but not displayed as a Focus Map After the successful generation of a Focus Map ZEN displays the notification The fundamental requirements as well as the strategy for usin...

Page 136: ...e imaging plane An image acquisition will be triggered automatically as soon as the system arrives at this Z position Next the system will travel back to the Z level it started from when was clicked T...

Page 137: ...the stage to the altered position The Step Size m display box show the currently used step size for stage movement The value can be edited by typing or using the arrowheads The Zero Position Set butto...

Page 138: ...ids should not be used together with Axio Imager and Axio Examiner equipped with a level adjustable sample holder During the calibration procedure the objective or neighbouring objectives will collide...

Page 139: ...into the sample holder until the clamps are clicking into place Fig 176 4 Life Science for TEM Grids Lift the spring Fig 176 5 of the appropriate position and turn it side wards Insert the TEM grid Fi...

Page 140: ...ly when you forgot to calibrate the holder before you acquired the image Choose general options for the calibration in the Marker Options field Move stage in load position before x y movement moves th...

Page 141: ...marker checkbox is activated If the stage is moving in the wrong direction invert the direction by clicking on X Invert or Y Invert in the Sample Holder Calibration field If the first marker position...

Page 142: ...on Restart Calibration in the Shuttle and Find tool window b Manual marker recognition If the marker on the sample holder is not recognized or user defined markers are used the marker positions can be...

Page 143: ...file format Enter the name of the image and click on Save It is possible to zoom the scan area using the Acquisition Mode tool but it is not possible to rotate or slide the scan area Mounting of the c...

Page 144: ...tion sample manipulation bleaching and image analysis for LSM imaging mode The control of the laser in LSM imaging mode via AOTF technology enables the restriction of the illumination to the defined r...

Page 145: ...Drawing of a circle in the Image Display window click and keep the mouse button pressed to set the center point drag the diameter let go off mouse button again to end the procedure Polyline Drawing o...

Page 146: ...choice of Color is inactive in this mode Common Color assigns the same color to all regions The Creation Mode drop down menu offers three different settings for defining ROIs Fig 191 Switch to select...

Page 147: ...currently displayed in the Regions tool in one file Saved images can be loaded later on by pressing the Load button using the Windows Explorer 5 2 15 Multidimensional Acquisition Z Stack The Z Stack...

Page 148: ...he Interval in a way that the total Range for the stack is maintained The Interval input box displays the z distance interval between slices in m This number can be edited by typing or using the arrow...

Page 149: ...e the focus position by using the slider tool right to the ruler Click on the circle symbol with the double arrow and move it up or downwards to change the focus This tool might also be used for defin...

Page 150: ...of Slices while adjusting the total Range The Slices number will remain unchanged Pressing the Optimal button sets Interval to match the Nyquist criteria 2 fold over sampling and accordingly sets the...

Page 151: ...s Position updates accordingly The Offset value then might be set to half of the Interval depending on the interval value and the number of slices as with a lower number of slices or a large interval...

Page 152: ...r position the Z Piezo stage can move half the total range each up and down Define the first and last slice using First Last mode by pressing the Set First and Set Last buttons at the respective focus...

Page 153: ...hannels detecting the longer wavelength range In case the channels are assigned to different tracks and a Frame wise Multitracking scheme is applied the pinhole diameter is set for each track such tha...

Page 154: ...e highlighted Position in the list without scanning Starting a Continuous scan then allows setting or correcting the parameters and storing them again hitting Add Highlighted positions can be removed...

Page 155: ...ion increase of the SR mode in Z a doubled sampling with smaller Z intervals is required This is done automatically when the SR mode is active Optimize Sectioning and Step Optimal In case the interval...

Page 156: ...the percentage value in the box next to Stop when Intensity drops to The number of Iterations indicates the total amount of scans which are performed for bleaching the selected region see also sectio...

Page 157: ...nd laser intensity which is then used for bleaching It is possible to choose different laser lines and or laser intensities for the bleach process of each region when activating the option Use differe...

Page 158: ...e the stage does not move exactly horizontal to the scan field a Rotation value can be set in the Rotation input box to correct for this available only in Show all mode This can either be calibrated b...

Page 159: ...n Windows explorer If Centered grid is selected the current position will be the center of the Tile Scan If Bounding grid is selected positions that should be part of the tile scan have to be marked u...

Page 160: ...will be displayed in Pixel and Size m units the objective from the Objective drop down menu and a potential zoom factor via the Zoom slider or Zoom input box with which to scan an overview image In t...

Page 161: ...ion mode Note that it is an upper limit of handling data files but this is beyond the value of 100 Gigabits Interval defines a potential time interval set in milliseconds ms seconds sec or minutes min...

Page 162: ...Time You can load a predefined timer configuration from the Interval Time field by pressing the Load button symbolized by the folder icon Fig 213 All saved timer will be listed in the Load configurati...

Page 163: ...o indicate the start or end of an external process in the data of the image series by connecting this external process to the selected Trigger In for that Marker Up to 4 different triggers can be sele...

Page 164: ...d marker configurations are displayed To search for a marker configuration type in its name or part of its name in the Search field and only those marker configurations will be listed that contain the...

Page 165: ...e comments are stored along with the image contents The markers visible in the image series have different colors with the following meaning red manually set marker with time indication and comments b...

Page 166: ...Scan checkbox is ticked Fig 228 a continuous scan is performed when clicking Start Experiment button but no image series is acquired until you hit the Go button which becomes visible next to the Pause...

Page 167: ...re the cycles have been processed the Time Series will only be interrupted Waiting for Trigger will be displayed in the status line The Time Series can now be continued via a new trigger signal or end...

Page 168: ...arameter Focus Devices and Strategy Highlighted positions can be removed by pressing the Remove button Alternatively the whole list can be deleted by pressing the Remove All button It is also possible...

Page 169: ...ier by clicking on Properties of the Carrier definition field By using the Columns and Rows Number input boxes you can define the numbers of columns and rows of the carrier In the Columns and Rows Dis...

Page 170: ...section where all saved carrier configurations are displayed To search for a carrier configuration type in its name or part of its name in the Search field and only those carrier configurations will b...

Page 171: ...this well of the plate This assignment will be kept until changed repeating this procedure and that well will turn blue Pressing the button Fig 243 will display a help text how to calibrate the posit...

Page 172: ...e context of the Positions tool and not in Focus Devices and Strategy If individual offset values are specified in position lists these values are ignored upon switching to an autofocus mode other tha...

Page 173: ...the LSM imaging mode a true zoom is taken For widefield modes only the zoomed in part of the camera chip will be displayed Changing Objective and Zoom will change the size but leaves Pixels unchanged...

Page 174: ...menu Fig 248 The scroll bar can be used to have access to all listed file formats In the Dimension selection field you can define how the image series is saved For each dimension you can define by ti...

Page 175: ...can even interfere with the timing of the acquisition 5 2 23 1 Requirements Position the specimen under the objective and focus onto the desired imaging plane Activate Show all Tools and open the Imag...

Page 176: ...up tool now displays the way the potential emission signal takes Channel 1 receives the red part of the spectrum The settings are suitable for imaging dyes like Rhodamin TRITC or Cy3 For overlaying fl...

Page 177: ...er default set to unidirectional The default settings for Acquisition Mode are suitable for good image quality and do not need to be changed for initial scanning 5 2 23 3 Defining the Detection Parame...

Page 178: ...se the Digital Offset slider so that areas without picture content just show a trace of blue If necessary re amplify brightness with the Digital Gain slider Stop scanning Save the imaging configuratio...

Page 179: ...fset are controlled as these are set individually for each channel Use the split view see section Split View to see the effect of the controls onto each channel separately If the acquisition is perfor...

Page 180: ...2014 V_01 Raw data sum image Range Indicator Fig 257 In order to fine tune the detection the raw sum image source image left of the Airyscan should be observed carefully Use the checkbox of the Range...

Page 181: ...Area If active all new data will be added to the document Old data can be added to the new document by drag and drop from stored documents Countrate action button Opens the Countrate window which dis...

Page 182: ...n area check boxes Activate certain toolbars in the display Laser tool Setup Control to switch on off lasers and check their status Light Path tool Setup Define store delete and load predefined light...

Page 183: ...se the tool The display will change for different configurations The following combinations are possible 1 32 GaAsP array one side PMT and optional 2 Channel Module BiG 2 and or NDD BiG 2 as NDD Fig 2...

Page 184: ...nked to the selected beam path and called up whenever that beam path is selected later 2 Loading a predefined beam path You can select a saved beam path configuration from the drop down menu The selec...

Page 185: ...light major beam splitter is not available Secondary dichroic beam splitter SBS The SBS splits the fluorescence spectrum onto the various detection channels The long pass LP filter lets wavelengths p...

Page 186: ...tabase is not updated the old filter names will stay To adjust the pinhole for this beam path press Adjust pinhole and proced as described below Selection of channels You can select channels by activa...

Page 187: ...visible light for UV 405 445 and NLO laser Secondary dichroic beam splitter SBS The SBS splits the fluorescence spectrum onto the various detection channels The long pass LP filter lets wavelengths pa...

Page 188: ...intain tab The spectral detection band is displayed and the type of filter is indicated in the tool tip for each detector BiG 2 can be combined with up to 4 NDD PMT channels The Light Path only shows...

Page 189: ...lection of dichroics and filters From top to bottom you have the following dichroics and filters available Main dichroic beam splitter MBS The MBS reflects the specified laser line and allows the resu...

Page 190: ...measurements and pinhole adjustments Lasers area or bleaching Bleach area Fig 266 Press again to close the panel Activate the lasers you want to use by checking the corresponding boxes The lasers wit...

Page 191: ...um value is obtained The correction ring should also be used for adjusting the Counts Molecule setting whenever the immersion media is changed This is especially important in cases where the refractiv...

Page 192: ...The 1 AU button allows you to set the pinhole size to 1 Airy unit by just clicking on it Set the desired pinhole diameter using the slider the scroll arrows or type into the input box This will alter...

Page 193: ...ustment in x and y by pressing the Fine button The pinhole will travel only a limited distance around the peak found in the coarse alignment If the pinholes are adjusted for the first time the coarse...

Page 194: ...ows If both coordinates are changed the system will regard this as a new alignment The pinhole does not move in z The collimator for invisible light is set to the optimum position in the factory and c...

Page 195: ...collimator in the Collimator panel for invisible light To find the optimum collimator settings use either the count rate or images This function should be changed with care only The optimal collimato...

Page 196: ...as one 100 sec measurement If bleaching becomes a problem it might help to reduce measurement time and increase repetitions to obtain nearly as good statistical results while reducing bleaching Bleach...

Page 197: ...and two cross correlation channels With the 32 Channel Module you have up to eight auto correlation channels and accordingly 8 faculty cross correlation channels In the Lasers area you can activate an...

Page 198: ...table The table has the following entries Fit range start Designates the start value of the correlation function taken into account for the fit You can change the value by typing in the display box Fi...

Page 199: ...to 6 the system will report this conflict Before saving you can return to the old default settings by pressing the Reset button This button is also available from the Fit parameters window which can b...

Page 200: ...b again to close the panel You have three options to define a model Correlation assemble a correlation model from predefined equations which will be fitted analytically PCH assemble a photon counting...

Page 201: ...displayed in the G t 1 display area The name of the model will be displayed in the Model display box You can load a pre defined model from the Model drop down menu Its name will than be displayed in...

Page 202: ...ssible to improve on the fit quality It does not make too much sense to fit to three components without fixing parameters of at least one of them For example if the diffusion time of a free ligand can...

Page 203: ...ters For some equations useful conversions to other parameters are listed as well The total correlation is given by equation 2 k l l k I tot G A B d G t t 1 2 where d is the offset B the background co...

Page 204: ...fixed value In some cases especially if very slow or immobile components are present there can be a positive offset from 1 This offset can be taken into account by fitting to d On the other hand if t...

Page 205: ...ylindrical 2DG 0 500 2 D Gaussian 3DG 0 350 3 D Gaussian GL 0 076 Gaussian Lorentzian can also be calibrated if a known concentration c of a dye is measured In this case N can be fixed and fitted The...

Page 206: ...calculated from equation 5g N can have different meanings in different fit models For biology normally the number of diffusing particles is of interest In this case if photo physical processes triplet...

Page 207: ...ferred to as the lifetime B 2 Dependent in combination with other terms a e C Ga 6c C in this case is either a fit parameter or a fixed value and often takes the value 9 5 There are two cases to be di...

Page 208: ...d exponential anti bunching This is a double exponential function where the exponentials are subtracted 2 2 2 1 1 1 2 2 1 1 1 e K K e K K G not normalized 6f 2 1 2 1 1 1 2 2 2 1 1 1 K K e K e K t G k...

Page 209: ...parameters Triplet is based on an un allowed intersystem crossing from the excited to the so called triplet state This state lasts for 1 5 s If the electron drops back to the ground state no photon i...

Page 210: ...pecies D or B in Hz or the relative dimensionless brightness In case where darker state is completely dark D 0 equation 7h simplifies to R F R b k k k T 7i Note that Blinking is referred to a process...

Page 211: ...are the fractions of molecules in the triplet state and tt1 and tt2 the triplet exponential decay times T1 T2 tt1 and tt2 are all fitted parameters C 5 Stretched exponential bunching In some reaction...

Page 212: ...2 1 2 1 1 1 2 2 2 1 1 1 K K e K e K t G k k k k k k k t t t t normalized 7q where K1 and K2 are the fractions of molecules and tk1 and tk2 the exponential decay times k1 and k2 the frequency factors a...

Page 213: ...y factor and tr m the rotational diffusion time However there are special cases that are of more use In symmetric rotation the general formula reduces to r e R R G a a r 1 not normalized 8b a a r R e...

Page 214: ...l diffusion times Ra tr 1 and tr 2 are fitted parameters c1 and c2 as well as r1 and r2 are fixed values and must be user defined Rotational frequencies take often the following values r1 1 r2 10 3 Th...

Page 215: ...1 ed2 1 ed1 and ed2 are fixed values and have to be user defined The following values define 1 2 and 3 D diffusion ed1 ed2 dimensionality 1 2 0 1 D 1 0 2 D 1 1 3 D Note that in the FCS software these...

Page 216: ...th representing the transport coefficient of the fractional time dimension Please note the following relation between D and G 1 t t D 8j If activating the fitting to the diffusion coefficient in the F...

Page 217: ...rue fraction fi of each species values those can be retrieved with the known brightness from the relation 3 1 2 2 i i i i i i f f with the constraints and 8n If there is no brightness difference betwe...

Page 218: ...f 8p In the presence of translational diffusion the term alters to 1 2 d f e Gf 8q with tf representing the average residence time for flow and td the diffusion correlation time Note in the FCS softw...

Page 219: ...rrelation terms for kinetics All highlighted terms are assembled into the final equation that is displayed in G t display area For a selected term the Settings button becomes available Press the Setti...

Page 220: ...ou can set a value for the geometric factor which describes the point spread function Fig 278 By pressing the cylindrical 2DG 3DG and GL buttons you can select a cylindrical 2 dimensional Gaussian 3 d...

Page 221: ...on and translation form by checking the respective box This equation will be used as an additive term to rotational and translational diffusion terms You have the option to leave the amplitude value f...

Page 222: ...e set for the relaxation times noise might also be followed and triplet fractions might show up to be too high If weights are applied noise is followed less The following weight equation is used 6 10...

Page 223: ...Dependent antibunching rotation and translation by checking the respective box This equation will be used as an additive term to anti bunching and translational diffusion terms You have the option to...

Page 224: ...d by selecting the excitation polarization linear polarized or unpolarized and the emission detection parallel perpendicular or all by pressing the corresponding tabs which will result in the correspo...

Page 225: ...in the r selection box Either type in a value or use the arrow keys You must also specify if you use two photon excitation by checking the 2 Photon check box if since that will influence the fit formu...

Page 226: ...heck box Fig 287 In the latter case you have to provide the radial dimension of the confocal volume in the r selection box Type in the number or use the arrow keys The description box gives you inform...

Page 227: ...on normalized terms by selecting deselecting the Normalized check box You can select between dependent and independent stretched exponentials by choosing from the Components drop down menu This become...

Page 228: ...r each activated component you can enter a brightness value in Hz in the Specific brightness enter boxes If you don t know the brightness enter 1 You can enter Instrumental parameters values in the re...

Page 229: ...ables are defined you have to finish with the return key Each defined variable must have an assigned number This number will substitute for the variable in the equation A formula has always to be star...

Page 230: ...ich will be identical to minimal bin time The Maximum correlation time defines the end correlation time It cannot exceed the measurement time Hence the value will only be used if equal to or smaller t...

Page 231: ...292 Click any other tab to leave the panel You can either check the Automatic check box in which case dynamic binning will be applied or you can de activate the check box and enter a number of a cons...

Page 232: ...ected and displayed In automatic binning mode the Binning display window and the 10 default button will not be accessible 4 Dust filter The Dust filter panel allows you to activate an electronic dust...

Page 233: ...intervals before and after the discarded region are separately correlated and the results averaged These holds also true if more than one region is discarded In this case all the single regions that...

Page 234: ...to this defined position Further clicks will move the stage in the direction of the arrow pressed with the step size and speed selected You can stop the stage from moving to a defined position by pre...

Page 235: ...w configuration by pressing the Save button floppy disk symbol This will open the Save current carrier configuration as dialog Fig 300 Type in the Configuration name you want to use Press Ok to save t...

Page 236: ...between the chambers in the appropriate Column and Row input boxes To orient the sample carrier in x and y click on the Calibrate Position the center of one chamber over the center of the objective b...

Page 237: ...z position to zero If a z Piezo stage insert is available the Piezo field will be displayed The Piezo Position m box displays the actual z position of the Piezo stage insert The arrows will move the f...

Page 238: ...king on one well in the X Y Stage panel of the Sample Carrier tool Set up a beam path in the LSM Imaging Setup tool Fig 305 Convenient settings are 488 nm line at 50 tube current and 5 AOTF MBS T80 R2...

Page 239: ...LSM Acquisition Mode tool Fig 306 Choose Line scan highest Scan speed 512 pixels and Zoom 1 for convenience Set up scan control in the LSM Channels tool Fig 307 Open Pinhole to maximum Set detector ga...

Page 240: ...u might adjust the line to lower or higher values by altering the gain or laser power Move the objective cautiously upward by turning the Z focus knob of the microscope stand A lens moving upward will...

Page 241: ...ximum Fig 312 which corresponds to the upper cover slip surface Store the reached position by using the Store button Now you can position the detection volume into the sample in a well defined way If...

Page 242: ...hed the system will take the minimal required time i e it completes the data point and immediately starts the next Shape allows you to choose from three possibilities of distributing the time spacing...

Page 243: ...period between two successive measuring cycles as specified in the Point to Point time selection box is increased by an exponential factor of which the initial base is defined in the Factor selection...

Page 244: ...andard positioning Step 1 slow speed should only be used for very precise positioning since positioning for longer paths requires more time Step 3 is suitable if large vessels are used since precise c...

Page 245: ...has been done this position corresponds to the middle of the image In other cases the position of the laser beam with a panning within the image has to be determined by a bleach experiment You can us...

Page 246: ...in the Properties drop down menu You can save the carrier delete a saved one or load a predefined one with the appropriate controls Selection of a sample carrier In the Properties drop down menu box y...

Page 247: ...disc or cross buttons The selected chamber is then approached automatically via the motorized microscope stage When the defined position has been reached the position of the chamber is displayed in t...

Page 248: ...zed stage is available Otherwise the tab is not active Select one of the three speeds from the Speed Settings drop down box Select the step size from the Step Size m scroll menu or the input box with...

Page 249: ...ft Column by column Measurement is performed column by column Each column starts from the top Column by column meander Measurement is performed column by column Columns start alternatively from the to...

Page 250: ...ll go back to the initial position after the end of the measurement If you want to obtain the count rate by pressing the Count Rate button at the indicated position using the X Y Stage control you hav...

Page 251: ...d positions respectively The measurement procedure starts and the Image FCS diagram window appear in a container 4 Hysteresis correction of Scanning mirrors In general there is no need to compensate b...

Page 252: ...tton The system will adjust automatically the Scan Corr X and Y values Alternatively you can do the compensation manually If the Scan Corr is not adjusted you will see a pixel shift Fig 329 Alter the...

Page 253: ...desired Channel The numbers on the right will display the actual temperature If a sensor is available its temperature will also be displayed The Atmosphere panel allows defining the O2 and CO2 concen...

Page 254: ...data Fig 332 Click a second time to close the information window The scroll bar can be used to view the complete information The Copy button will copy the content of the Information On Experiment win...

Page 255: ...ample Carrier or highlighted LSM Image position Start Experiment Starts sequential measurements on all marked or highlighted positions Stop Ends or interrupts the current measurement Appears only when...

Page 256: ...onfirm overwriting the old method Press Yes to overwrite No to keep the old method settings If the system settings deviating from default expectations it will give a warning For example if no pinhole...

Page 257: ...panel lists all existing measure methods for selection or deletion You can select the analysis method you want to use by highlighting the corresponding entry from the list with a single click and pre...

Page 258: ...ow will appear Fig 336 To search a configuration enter the respective name in the Search display field If there is no match with the Configurations list no method will be highlighted You can also choo...

Page 259: ...ement is triggered by pressing the Snap or Start Experiment button and no active FCS diagram window is open in the Container a new window will be opened displaying the count race trace correlation fun...

Page 260: ...alue is constantly updated and calculated each 0 5 ms if Averaging is switched Off If system overload occurs an error message will be displayed and the APDs switched off in order to avoid damage Fig 3...

Page 261: ...el can be closed by the Channel 1 and Channel 2 arrows b Unit Select the units of the Count rate and CPM displays in Hz or kHz by toggling between the corresponding buttons c Average Averaging for 1 s...

Page 262: ...the active scan direction Define the extent of the scan by using the Start Position m and End position m input boxes or their scroll arrows You can see the current position in then Current Position m...

Page 263: ...l black line line plot or the cross lines frame plot can be used to re position the focus Just drag the bar or cross lines with the left mouse button held down to the new position and release the mous...

Page 264: ...in the z scan range it will also give a peak To determine its position just perform a z scan at a position where is no cell If the glass reflection contribute so much that the peaks of the membranes...

Page 265: ...of interest at the site of the fixed laser beam in Current Position using the Crosshair or use the crossline in the LSM image to mark the position In the latter case Scanner must be active since the...

Page 266: ...o save any changes No to discard the alterations and Cancel to return to the diagram Fig 347 A scrollbar at the bottom of the window will allow you to view all data in case the content does not fit th...

Page 267: ...count race trace correlation function photon counting histogram and pulse density histogram It also displays the result table Coincidence Displays the coincidence diagram of a two channel measurement...

Page 268: ...sing the Snap or Start Experiment button After the measurement you can store the data and open it for later use Stored data will be loaded in an extra FCS diagram in the specified container If you clo...

Page 269: ...ement at the first Carrier or highlighted LSM Image Fig 351 position will be performed If Start was selected measurements will be performed on all selected positions in a sequence Status lines at the...

Page 270: ...they can be imaged with the LSM Alternatively suitable cells can be searched by using low zoom values in the LSM Visual inspection of the cells via binoculars In the main toolbar activate the Locate t...

Page 271: ...the stage in the direction of the arrowheads Fast movement is obtained with the outer rose slow movement with the inner rose The X Position m and Y Position m display boxes show the actual x and y coo...

Page 272: ...e highlighted in color For transmitted light observation Switch the HAL lamp on by the HAL On Off button and adjust the light intensity Activate the condenser function with the Condenser button Press...

Page 273: ...the extension you need and select the file you want to open Data formats that will be recognized in are fcs and raw Raw and FCS data files raw fcs will load together in a diagram if selected together...

Page 274: ...Rate Correlation Function G t Pulse Density Histogram Photon Counting Histogram and Pulse Distance Histogram It also displays the Result Table A scrollbar at the bottom of the diagram allows to view...

Page 275: ...the required position When the mouse button is released the column is inserted in the new position A scrollbar at the bottom of the table allows one to view all parameters that might not fit within th...

Page 276: ...all positions All rows belonging to the same measurement position than the highlighted row will be selected Select all kinetic indices All rows belonging to the same kinetic time point than the highl...

Page 277: ...ata sets Fig 361 submenu window Select the color you want to assign and press Ok All selected curves will be displayed in the color If you do not want to change color press Cancel 5 3 21 4 Image View...

Page 278: ...selected in the original table All other measurements will be opened unchanged To select all measurements use the table context menu opened with a right mouse click in the table and choose the line Se...

Page 279: ...her The hierarchy of grouping of categories is determined by their listed sequence from the drop down menu If the Show standard deviation box is checked all parameters will be displayed with their val...

Page 280: ...ers The cut out region is thereby displayed as a matted box Fig 295 You can select independent cut regions for different channels of a cross correlation experiments In cross correlation calculations a...

Page 281: ...rams via the clipboard The Paste function permits direct insertion of these data into the required program Select the line Write text to file to store the data in an external ASCII file txt Normalized...

Page 282: ...ER 1 SYSTEM OPERATION ZEISS Left Tool Area and Hardware Control Tools LSM 880 276 000000 2071 464 10 2014 V_01 Fig 368 The Correlation Curve window in normal upper graph and normalized lower graph dis...

Page 283: ...logarithmic scaling The line width allows you to select the thickness of the graph 6 settings are possible 4 Photon counting histogram This diagram also called Photon Counting Histogram shows the freq...

Page 284: ...itting results fit parameters raw data to a disk If Save as is pressed you will have the choice between FCS data files fcs without raw data or FCS files with raw data fcs attached by selecting the app...

Page 285: ...program In the Export window you can enter a name in the File name field and a type in the Data type field In the Save in pull down menu you can select a directory You can specify if only the selected...

Page 286: ...unt rates occurring simultaneously in both channels will populate the diagonal of the diagram All the table functions in the Coincidence mode are identical to those in the Correlation mode The Coincid...

Page 287: ...s The panel is divided in Diagrams Result Table Model Field and Fit action buttons 1 Diagrams Fig 375 Fit diagram panel for correlation analysis For fitting to a correlation model the upper graph Fig...

Page 288: ...llows to select the line width for the selected graph Fig 376 Fit diagram panel for PCH analysis The Zoom function of the Fit diagrams can be used to display certain diagram areas of interest in an en...

Page 289: ...board with a click of the mouse if you want to insert the data directly into other WINDOWS programs via the clipboard The Paste function permits direct insertion of these data into the required progra...

Page 290: ...on diagram Start Channel and End Channel determine which part of the correlation curve should be fitted to the model The start and end position of the channels will be displayed as correlation times i...

Page 291: ...Copy data to clipboard with a click of the mouse if you want to insert the data directly into other WINDOWS programs via the clipboard The Paste function permits direct insertion of these data into th...

Page 292: ...ols LSM 880 286 000000 2071 464 10 2014 V_01 Lower and upper limits are only displayed if selected To select click with the right mouse button into the result table to open the context menu Fig 382 Fi...

Page 293: ...onging to one term are listed as a block in the Parameter column which shows the term and parameter names The current parameter values are displayed in the Values column You can edit a value by typing...

Page 294: ...t measurements to the same value All repetitions regardless of being selected or not in the result table will be taken for global fitting If you want to exclude measurements from the global fit they h...

Page 295: ...l perform a fit for all measurements rows that have the same channel as the one displayed in the Channel display field All other channels will not be fitted If you change to a row with a different cha...

Page 296: ...tarts the print function Printer Setup Opens the printer setup window to set the print parameters 1 Amount Compressed Selects the Compressed Report preview without diagrams Standard Selects the Standa...

Page 297: ...CHAPTER 1 SYSTEM OPERATION LSM 880 Left Tool Area and Hardware Control Tools ZEISS 10 2014 V_01 000000 2071 464 291 Fig 387 Print panel...

Page 298: ...Information panel The information panel Fig 388 contains three blocks Name Comment and Acquisition Names This is an entry box where you can input the name of the experimental set up Comments This is a...

Page 299: ...r must be used to have access to all functions The Processing tool provides a number of methods for image processing and analysis using mathematical operators and algorithms which include Adjust Airys...

Page 300: ...ALM and Channel Alignment Processing tools are available which can recognize tables If you want to apply other Processing tools the PALM image based on the table has to be converted to a true image Su...

Page 301: ...placed by another one This holds also true if the Processing tool is changed the previous loaded image will stay selected If another image is to be loaded just highlight that image in the image contai...

Page 302: ...mage 2 which can be selected by pressing the respective thumbnail on the left and the Preview image on the right In addition a new View tab the Preview tab is opened for the image in the Center Screen...

Page 303: ...ider or editing box to set a new value Fig 393 2D processing is available for 2D and 3D data sets also over time 3D processing is available for 3D data sets Z Stacks also over time only To process Air...

Page 304: ...e series or Z Stack will be processed Note that with 3D data you have the choice of a 2D or a 3D processing by selecting the method from the dropdown list Fig 395 A 3D processing will take longer but...

Page 305: ...After confirming your selection the batch processing will start and might take a few minutes to hours depending on the number and type of files selected All files will be processed with the same proc...

Page 306: ...Projection Processing tool select it from the list in the Processing main tab Click on the triangle buttons to show or hide the Method Parameters and Preview panels of the function Select the Input Im...

Page 307: ...sing tool select it from the list in the Processing main tab Click on the triangle buttons to show or hide the Method Parameters and Preview panels of the function Select the color gradient by choosin...

Page 308: ...isplay as Input Image and Input Image 2 The Output Image is automatically fed into a new image document A specific input channel from a multi channel image or series can be selected using the pull dow...

Page 309: ...Intensity range 0 1 should be marked This normalizes the image intensity for all images to values between 0 and 1 All actions performed in the Image Calculator have immediate effect on the previews Af...

Page 310: ...ect how many pixels you want to bin in x direction y direction and time points by setting the respective values in the X Average Pixels Y Average Pixels Z Average Pixels and Time Average Pixels input...

Page 311: ...he filter of your choice from the Type pull down Median Smooth Sharpen Band and Gradient Select the channels you want to process with the All Ch T1 or Ch T2 buttons Once a filter has been selected fro...

Page 312: ...pixels set by the Strength slider It has an analogous effect as the kernel size Fig 405 If a dimension should be processed by this tool the respective X Y Z and Channels check boxes must be ticked The...

Page 313: ...dimension should be processed by this tool the respective X Y Z and Channels check boxes must be ticked Gradient Filter The Gradient Filter visualizes the direction and rate of brightness changes from...

Page 314: ...y pixel by pixel image analysis procedure Experimentally fluorescence spectra of mono labeled samples are acquired and stored in the Spectra Database as an external reference Then a multi channel imag...

Page 315: ...acks only available in LSM imaging modes or multi channel images can be loaded In the Linear unmixing panel Fig 410 the number of spectrally distinguishable fluorescent components within the imaged sa...

Page 316: ...e following additional settings and functions are available for linear unmixing Auto scale balances the intensity of the unmixed channels to equal levels Display Channel with Residuals generates an ad...

Page 317: ...els with statistical confidence This option displays an additional channel per unmixed component which shows the relative statistical error in each unmixed component channel This statistical uncertain...

Page 318: ...ing signals this method will yield poor results Avoid saturation of fluorescence signal in the data set to be unmixed Saturation will generate a high signal in the residual channel To get the best unm...

Page 319: ...entrations in physiological experiments To open the Ion Concentration tools click Ion concentration Fig 414 The two buttons select the active image in the Image Display as Input Image and Input Image...

Page 320: ...the calibration method and if the calibration is carried out in vitro situ Ch 1 and 2 select the channels from Input Image 1 From these pull downs the following combinations of parameters can be chos...

Page 321: ...1 Single Wavelength Dyes Offline Calibration Subtract background autofluorescence image from raw images to obtain better raw data to start with Fig 417 Perform equation or titration calibration compar...

Page 322: ...ofluorescence over time Calculate absolute ion concentrations pixel by pixel via titration calibration known ion concentrations applied to the cells in situ or in solutions in vitro or equation calibr...

Page 323: ...ckground autofluorescence images from raw images to obtain Rkorr F1 F1Background F2 F2Background when calibration reference is not obtained with the experimental sample in situ Calculate ratio R Perfo...

Page 324: ...aturated with Ca2 Fmin2 and Fmax2 are the minimum and maximum fluorescence intensities at wavelength 2 Rmin Rmax Fmin2 and Fmax2 may be determined in the cells under investigation in situ or in soluti...

Page 325: ...ng mode or temporal correlation of an image or image stack To select an image highlight the image in the image container and press the Select button This will be your Input image As an output image th...

Page 326: ...image series Fig 422 Depending on the input image for example Z Stack or Time Series the available modifications vary which are available in the Modify mode drop down menu Fig 423 and Fig 424 It allow...

Page 327: ...the input meets the requirements for HDR imaging See full description of tool for details Please see also the description of the HDR High Dynamic Range function part of the Acquisition Mode tool for...

Page 328: ...bove the dynamic range Result divided by 1 Image x 2 cut off of grey levels above the dynamic range Result divided by 2 Image x 3 cut off of grey levels above the dynamic range Result divided by 3 The...

Page 329: ...display This step visibly intensifies weak signals Fig 427 Unlike the Sampling method the Illumination method of HDR still provides a linear grey stepping which allows quantitative measurements For t...

Page 330: ...e regions which are excluded from a second and or third illumination step are divided by 2 instead of 3 After acquisition the grey dynamics of the resulting image are not automatically compressed to m...

Page 331: ...led images acquired with Versions of ZEN 2012 and upwards and the tiling function can be stitched together in 2D and 3D Select a tiled image or stack with the Select button Fig 430 The Correlation Thr...

Page 332: ...Fig 431 is applied to data acquired in the PALM imaging mode It subdivides in 5 subcategories Convert to Image Converts the table vector map of a PALM image to a bona fide image Import PALM molecules...

Page 333: ...ert to Image processing tool Fig 432 Highlight the PALM image in the image container you want to convert and press Select Fig 433 Press Apply to convert the HR channel to a true image The new file has...

Page 334: ...ab to select the Import PALM molecules processing tool Fig 434 Press Select Fig 435 to open Windows Explorer and select the molecule table you want to import Press Open to load the table and then pres...

Page 335: ...calization precision will be estimated using a Monte Carlo Simulation of the experimental PSF including noise The generated lookup table will then contain the localization precision depending on the t...

Page 336: ...ettings Reuse button will recall the parameter setings of the actively selected image The Channel drop down menu allows you in a multi channel experiment to apply different settings to different chann...

Page 337: ...d due to the Maximum cluster size setting The Dimension drop down menu will display the dimensionality 2D or 3D of the selected PALM image which is automatically recognized if a czi format is loaded F...

Page 338: ...the circles will correspond to the set value of the Peak mask size Fig 448 The bigger the radius the more prone are closely juxtaposed peaks to overlap The smaller the circles the more likely it is t...

Page 339: ...single and overlapping peaks will be taken into account However fit accuracy will be decreased when multi emitter events are fitted as the distribution of one emitter will influence the distribution...

Page 340: ...eak is cut out of the frame and the next highest intensity peak is selected The procedure is repeated until I M SD x PI Than the peak finding process will end for that frame All identified peaks above...

Page 341: ...calculated PSF based on the objective lens and laser line used Note that with the Mask Fit only one predefined PSF width will be used Computation of the Fit will be faster for the Mask Fit but less pr...

Page 342: ...box must be ticked Note that for lateral drift correction the system needs to identify at least one fiducial marker based on the Drift Correction filter settings Fiducial markers could be gold partic...

Page 343: ...ot influence the Preview image Note that using Grouping in the processing tool is irreversible Therefore in the created PAL Grouping tab all functions will be greyed out and not accessible If all para...

Page 344: ...reshold If the number of counted neighboring peaks molecules falls short of the set value the respective peak molecule will be removed from the list and the PALM image This tool will emphasize densely...

Page 345: ...e Method Parameters tool window to have access to the Particle Tracking tools Fig 465 You can set different parameters to define the molecules and the calculation of the trajectories Peak intensity to...

Page 346: ...340 000000 2071 464 10 2014 V_01 Once the settings are complete press Apply to obtain the trajectories of the single molecules Fig 466 Please note that Particle Tracking only works for 2D data sets Fi...

Page 347: ...a 3D stack of a widefield image or a non processed raw data SIM image LSM image formats are not accepted to determine the Experimental PSF You have the possibility to automaticaly compute the Experim...

Page 348: ...s 7 times smaller as in the original image times Fig 471 The 3D PALM box will be automatically checked if the image data was acquired with the 3D PALM slider inserted In this case the double lobe stru...

Page 349: ...n two sorted lists of beads The beads that are closer to the medians in both orderings are assumed to have higher quality The Beads quality defines how far the beads characteristics intensity and size...

Page 350: ...sults for an Experimental PSF for 3D data will be obtained if beads are seeded sparsely for example 10 20 beads evenly distributed in a 512 x 512 image If Apply is pressed an average PSF from all the...

Page 351: ...orm accepts input other than SIM data as well See full description of tool set for details The Processing Structured Illumination tool works exclusively for data acquired in the SIM imaging mode It su...

Page 352: ...setting parameters Fig 477 If the Log FT check box is inactive the result is the absolute value of the Fourier transform sqrt Real x Real Imag x Imag with Real and Imag being the real and imaginary p...

Page 353: ...ab when selecting the image to have aces to the Structured Illumination tools You will have no tools available is the directory Structured Illumination tab is selected If a region of interest ROI is d...

Page 354: ...k that should be processed Fig 483 When Single is selected the slider or spin input box can be used to define one frame in the stack that should be processed Fig 484 With the Mode drop down menu you h...

Page 355: ...nd parameters become accessible to be changed and the Manual selection field becomes accessible Fig 488 The Automatic mode is recommended for images with high modulation contrast In case the modulatio...

Page 356: ...artifact by the Noise or Wiener filter will be kept and an offset is added to the whole image so that the minimal negative intensity becomes zero intensity baseline shifted In Baseline Cut negative va...

Page 357: ...displayed with best brightness and contrast If you want to keep the original scaling for quantitative studies you have to check the Raw scale box In this case the orifginal dynamic range is kept You c...

Page 358: ...n for Input image 2 Then expand the Methods Parameters tool window to have access to setting parameters Fig 498 You can toggle between Input image 1 and 2 in the Preview tab window which also displays...

Page 359: ...ys all channels of the SIM Input Image Fig 500 You can now link the TIRF channel to one of the SIM channels by highlighting the latter Than the respective TIRF channel will be used to compute its z re...

Page 360: ...aberration between different channels of an image or between images Highlight the Channel Alignment field to have access to the alignment tools Fig 501 Activate the first image you want to process in...

Page 361: ...cessing Tool Copy Subset and independently aligned The situation is different for PALM images The high resolution HR and sum widefield SWF channels are also listed alternatively with the HR channels p...

Page 362: ...ou can set a ROI and then the structure within the ROI is used For HR PALM images at least three fiducials are expected Without fiducials no alignment will be performed in this case Parabolic is used...

Page 363: ...Time select your reference image for example the first image or the center image by choosing for the Start and End value the same number Fig 508 Press Apply to extract that image from the time series...

Page 364: ...g image will have a doubling in channels The first half represents the original data set the second half the drift corrected data set Fig 511 The number of channels depend on the number of channels of...

Page 365: ...Input image 2 no alignment will be performed Loaded images cannot be unloaded but replaced by other images in selecting the respective image in the image container and then pressing the Select button...

Page 366: ...e coefficient table can be assigned individually to a channel That channel will then be corrected exactly with the coefficients of that ID If no alignment should be performed do not align can be chose...

Page 367: ...ries Z Stacks Combinations of Time Series and Z Stacks Multiview experiments as well in combination with Time Series When a dataset has been acquired using Dual Side illumination the dataset will have...

Page 368: ...Left slider or spin box the area of the left illumination side is defined This is marked at the slider itself with a red line the spin box shows the pixel value of the y axis Furthermore the cut off...

Page 369: ..._01 000000 2071 464 363 After pressing Apply a new czi file will be generated The original file name will be extended with _Dual Side Fusion For this resulting dataset the illumination dimension is no...

Page 370: ...e drop down list next to Algorithm to choose from the available deconvolution algorithms Nearest Neighbour The Nearest neighbor method uses the simplest and fastest algorithm Castleman K R Digital ima...

Page 371: ...cal sections It is also possible for missing information to be partially restored from neighboring voxels The spatial resolution can be increased without artifacts up to a theoretical limit one voxel...

Page 372: ...r images using an old fluorescent lamp that exhibits strong fluctuations in brightness Decay Corrects changes in brightness e g bleaching of the sample during acquisition of the Z Stack This function...

Page 373: ...Immersion input field displays the refractive index of the immersion medium Please note that this can never be smaller than the numerical aperture of the objective You can make a selection from typica...

Page 374: ...ence the Parameters interface The Registration will generate the transformation matrices so that all views are matched to overlay As a result a dataset is provided in which all Z Stacks of all views s...

Page 375: ...ton This will circle all elements recognized as beads in the chosen and displayed image frame Use the sliders in the Dimensions View control to change to a different view z slice and time point and pr...

Page 376: ...line the input box shows the pixel value along the y axis Furthermore the cut off is shown in the preview image with a red line for easy orientation It is best to set the illumination slider of the im...

Page 377: ...ve the same size If the scaling in Deconvolution PSF settings Fig 520 is changed the latter will override the scaling settings here Individual Registration For Every Time point If the multiview experi...

Page 378: ...performed Registration is then only done with the settings chosen in the Registration Method menu Hardware Rotation The Views are rotated solely based on the information from the rotation positioning...

Page 379: ...e image control area the Z Position Illumination side and View can be chosen In Fusion Subset in X with Preview the currently chosen View is displayed in the Current View box With the Left slider or i...

Page 380: ...experiment has been performed over time this option becomes available If activated each time point generates a transformation matrix and each time point is registered individually As a result the tim...

Page 381: ...the same size 5 4 19 2 2 4 Interactive Registration Interactive Registration allows to register the recoreded views of an experiment manually when the other alignment options fail for that dataset Fig...

Page 382: ...th which of the maximum intensity projections the process is started In case of using the Front View button the maximum intensity projection is calculated along the z axis and the x y axis are display...

Page 383: ...an proceed with the remaining settings for Multiview Processing If the data was acquired with dual side illumination which has not yet been fused the method to fuse both illumination sides is availabl...

Page 384: ...Left and Right as red and blue lines respectively Fig 532 To avoid a visible change in intensity along the two cut off lines for Mean Fusion with Fusion Subset in X an overlap of both illumination si...

Page 385: ...vailable If activated each time point generates a transformation matrix and each time point is registered individually As a result the time necessary for registration is multiplied by the number of ti...

Page 386: ...d for every dataset It is possible to define which portion of the Z stacks of the contributing views of the experiment shall be used for the fusion process To do so the checkbox Fusion Subset in Z mus...

Page 387: ...additionally formed in the same folder with the nomenclature Name time point 1 czi e g FishEmbryogensis 221 czi This master file must be selected in the Method Parameters window There the settings fo...

Page 388: ...multichannel images apply to the new image Copy Channel Copy Channel produces a copy of the selected image The first Select button in the selection panel selects the active image in the Image Display...

Page 389: ...are dropped out For example setting Step to 3 takes 1 and drops 2 pixels along the respective dimension reducing the data to 1 3 The scaling is calculated to remain correct in the resulting image Sub...

Page 390: ...sis and Remove structures Fig 540 5 4 21 1 Detrend The Detrend function is a high pass recursive filter Click Detrend in the ICS tool The Method Parameters menu will show the input options for this fu...

Page 391: ...ve m moving average frames with an overlap of m 1 and average these The outcome is a new stack with n m 1 gliding or moving average frames Calculate from each of the average frames the overall average...

Page 392: ...window The correlation to be computed can be selected from the Correlation drop down menu Fig 543 For one channel recordings only Spatial auto correlation S A is available for a two channel recording...

Page 393: ...e and the Mean value variance check boxes Note that both filters can be applied together by checking both boxes Thresholds for the filters can be entered in the corresponding input boxes by typing or...

Page 394: ...or of 3 but better by up to a factor of 10 A pixel size of about 30 nm is beneficial as the method needs to be able to detect the fluctuation of the molecules within the confocal volume The imaging sp...

Page 395: ...vements for example of cells When activated the trend moving average average over the whole time series will be subtracted from the input time series prior to N B analysis Number of Frames is the numb...

Page 396: ...Number image N and the Brightness image B or if selected the Number image n and the brightness image e Use the Coloc View Tab to display the result as a scatter plot where either the Number or Bright...

Page 397: ...5 4 22 1 Burn in Brightness and Contrast The Burn in brightness and contrast function creates a new image document in which the current brightness and contrast settings from the Display View Options...

Page 398: ...possible options Use the List size spin box to set the number of fixed data points The table in the Interpolation panel will be extended to the selected number of rows Highlight a row of your choice...

Page 399: ...splay as the Input Image The image has to be a multi channel image Select the channels you want to shift by ticking the Ch1 T1 or Ch2 T2 check box Use the X and Y sliders or spin boxes to define the s...

Page 400: ...ut image s The corrected image is displayed in the center screen area In addition the calculated reference image can be stored and used for further shading corrections of other images Therefore the ch...

Page 401: ...s field The tool tabs in the Maintain field allow the calibration and adjustment of hardware settings The following options are available Adjust Pinhole and Collimator for LSM imaging mode Define Obje...

Page 402: ...n can be called up in the Maintain panel in the Adjust Pinhole and Collimator tool of the Maintain tool group see Fig 554 The position of the pinhole X Y Z coordinates in relation to the detector make...

Page 403: ...pinhole in relation to the beam path Z position can be set only for PH1 using the slider or arrow buttons Status display for setting procedure green for ready and red for busy Stored Pos button Pinho...

Page 404: ...ield Display of the relevant active Collimator Position mm field Setting of collimator position using the slider or arrow buttons the display to the left of the slider indicates the current position S...

Page 405: ...image must be scanned by the continuous scan method Select a fast scanning speed Measurement with Average Number 1 only no averaging of several measurements On the Channels tool in the LSM Acquisitio...

Page 406: ...the Position mm slider in the Collimators panel allows the collimator to be adjusted to maximum image brightness Optimum collimator adjustment obtained in this way can be stored by clicking the Curren...

Page 407: ...nts listed in the Requirements for Adjustment window Fig 559 Press Cancel to abort the automatic pinhole adjustment Press the OK button to proceed Pinhole adjustment will then run automatically and th...

Page 408: ...hen an objective was added newly or replaced by another one without having to leave the software Once the objective is activated its focus speed and parfocality can be set 5 5 2 1 Objective Press the...

Page 409: ...ectives showing all the objectives that have been previously selected User Defined Objectives showing all the objectives that have been previously defined by an user Fig 562 or Potential Objectives sh...

Page 410: ...Enter the parameters of the new objective in the appropriate display boxes or drop down menus The text of those boxes that are inactive for editing are greyed Press the Apply Add button to store the...

Page 411: ...he objective from No to keep it in the database or Cancel to abort the process 5 5 2 5 Show Edit Objective This function allows viewing the parameters of an objective from all objective directories Ed...

Page 412: ...e Write button the new settings will apply for computer and manual control 5 5 2 7 Parfocal Correction Press the Parfocal Correction tab to have access to the Parfocal Correction definition window Fig...

Page 413: ...y becomes available when the Start button is pressed Press Next again until all objectives have been selected You can cancel the process any time by pressing the Cancel button After the last objective...

Page 414: ...ystem toggles between these two processes For image acquisition the status should be indicated as Good Waiting Adjustments will only be performed during continuous scans if activated or during delay t...

Page 415: ...to the System Options setting window Fig 577 The left and right pointing triangles give access to all functions by sliding the tabs to the left and right directions The following user accessible prog...

Page 416: ...guration is saved and loaded The following parameters are available Objective Frame size Speed Data depth Scan direction Average Pinhole diameter Detector gain and offset Zoom Rotation and offset 5 5...

Page 417: ...or depth into grey values The Z Stack Waitstate function delays the image acquisition during time series or Z Stacks in case vibrations of the stage occur and are registered at the beginning of indivi...

Page 418: ...to open all files including the master file as a separate file Master files are generated when images are saved in individual files along time point stage position tile Z stack position and or channel...

Page 419: ...the specified file upon closing the Image FCS diagram You will be prompted in the Save raw data dialog box if you want to save the raw data Press Yes to save the raw data permanently along with the fc...

Page 420: ...the fcs file no manipulations will be possible in this case You can open raw data in the File menu of the Main tool bar You can either open the whole fcs measurement in which case all raw data will be...

Page 421: ...ucture of the recorded word Two examples should make the format clear Fig 585 The tables show part of the running pulse train from left to right divided into clock cycles 1 in the corresponding box in...

Page 422: ...the raw data in file offset hexadecimal and as ASCII Fig 586 Rel 4 x and ZEN raw data format The first 64 bytes bytes 0 63 represent the file identifier with the channel number The next 16 bytes bytes...

Page 423: ...imeters Only integer numbers of millimeters are possible Type into the Distance x and Distance y display boxes or use the arrowheads Speed of the stage Only the three preset stage speeds 0 48 mm s 4 8...

Page 424: ...ight 5 5 6 3 FCS Options Auto Save The Auto Save panel Fig 588 enables you to instruct the program to save the results automatically and continuously during the measurement process All data recorded u...

Page 425: ...ional Concept of the Center Screen Area and the Image Display Container 6 1 1 General Structure In this section the Center Screen Area of the ZEN Main Application window is described The Center Screen...

Page 426: ...sly activated Image tab the image document remembers which view type was activated before and displays the same parameters upon return to this image Image tabs The Image tabs show every opened window...

Page 427: ...tion There are two groups of View Option control tabs A general one which contains the blocks Dimensions Display Player and Graphics The second group consists of View tab specific tool tabs hosting to...

Page 428: ...in section Processing Copy Image Handling and Container Layout Configuration The control area Fig 594 on the top right corner of the Image Display is for scrolling click on the left or right triangle...

Page 429: ...utomatic container layout choosing between separate or shared view controls between the containers The same menu is available in the menu bar in the View part of the menu Splitting the Center Screen A...

Page 430: ...dimensions of the image data set The sections slices in the dimensions can be scrolled with sliders Z Position and Time inFig 598 and also directly addressed with setting numbers in the spin boxes ne...

Page 431: ...window Interpolation check box activates deactivates the zoom interpolation function 2 Dimensions Channels The Channels functions are designed to switch on off channels or the display of the merged i...

Page 432: ...sition Mode Click on will display the Crop Rectangle in the Image Display Fig 602 Any changes done with the Crop Rectangle are setting the scan parameters immediately On the next execution of a scan S...

Page 433: ...ge using the button Clicking on the button sets the intensity minimum to Black and percentile value and the intensity maximum to White and percentile value Activating the check box sets the intensity...

Page 434: ...setting of the Start sliders limits the number of slices to be used for the animation Slices before Start and after End are not taken into animated These sliders can be changed during automatic animat...

Page 435: ...on e g the z position in a Z Stack or the timestamp in a time series Functional Description The graphics function uses a plane separate from the image plane the graphics plane and therefore does not c...

Page 436: ...ated during image acquisition and can be exported with the image The annotation can be stamped into already existing images Scale bar Creation of a horizontal or vertical scale bar with default increm...

Page 437: ...ontour or volume Coordinate and dimension display shows the XY coordinates of the object center and the width and height of the graphic element Function elements of the graphics list box Activate deac...

Page 438: ...ged in the display by changing settings in the Display View Option control block or for example starting the Player animation This feature is particularly useful to display time or z position in an ex...

Page 439: ...options control blocks apply with the following additional features The Dimensions View Options control block shows the Merged tick box to activate deactivate the display of the channel overlay and a...

Page 440: ...ig 610 Channels if not rearranged by the user are by default displayed by their color that is the different modes are listed together If the Software recognizes such mixed mode channels the mixed mode...

Page 441: ...nnels belonging to the same color will be merged in one image Fig 613 If group modes is selected all channels belonging to one mode will be merged in one image Fig 614 Fig 613 Image Display Split view...

Page 442: ...of images from the original stack and store the result as a new image controls for this function are in the Processing tab function Copy Subset see section Processing Copy The settings of the Dimensio...

Page 443: ...w Option Tab Dimensions All Airyscan display and online processing functions can be done in the Airyscan tab Fig 616 The tab shows both the Airyscan source image and an online processing image side by...

Page 444: ...curves histogram of the source image and the processing preview are very different To get a balanced view on both images the display settings are adjusted automatically and can be individually optimi...

Page 445: ...gives a real time impression of the 2D processing result Fig 620 This can be both the SR mode and the VP mode You can see the improved resolution or the modified sectioning this way Deselecting the ch...

Page 446: ...available in VP mode To get optimal adjustment in VP mode it is recommended to switch to SR mode from time to time and do a continuous scan Auto In case the Auto checkbox Fig 622 is engaged the proces...

Page 447: ...ffective PSF of each element plus added noise equation 1 In the frequency domain the Fourier transform FT of the image of each detector element will be the product of the object spectrum with the effe...

Page 448: ...th the Wiener filter which can be set by moving the processing slider those frequencies which are otherwise not detectable due to the bad signal to noise ratio can now be emphasized The filter evens o...

Page 449: ...hree dimensions The settings of the Dimensions Display Player and Graphics view options control blocks apply In addition to the 4 general View Option control blocks the view specific Ortho View Option...

Page 450: ...ioning this symbol with the mouse you can move the XZ and YZ section planes to any point of intersection with the XY plane A click with the left mouse button places the intersection to the desired pos...

Page 451: ...the button to set the first XYZ point for the measurement of the spatial distance Set the second XYZ point for measurement by moving the X Y Z sliders or by moving the green red and blue lines in the...

Page 452: ...he Dimensions Display Player and Graphics View Options control blocks apply Any changes done with this toolbar are effective immediately The content of the overlay plane is temporarily hidden while th...

Page 453: ...w Options control blocks apply The settings of Display Player and Graphics View Options control blocks do not apply Any changes done with this toolbar are effective immediately The content of the over...

Page 454: ...tab contains the following function elements Profile button Profile display vertical polygon display Setting of the Profile Distance between 1 and 20 using the slider Grid button Grid display horizont...

Page 455: ...N Module 3D VisArt The LUT for the channels set in the View option tab Dimensions and interpolation settings as well as the parameters set in the View option tab Display are applied All other paramete...

Page 456: ...layed and can be operated individually via 3 Buttons The Cut button toggles between different clipping behaviors of the respective planes On the Clipping Planes different surfaces can be chosen from a...

Page 457: ...lluminated by a virtual light source The volume is viewed as if looking through the eyepieces of the microscope and the light source projects a shadow onto a background plane This gives the data a ref...

Page 458: ...reconstruction image in a shadow projection where the viewing point can be defined In addition to the zoom setting the image can be rotated around the three orthogonal axes via the relevant setting wh...

Page 459: ...n blue and the Z axis in green A click on the Scale button display an X Y and Z scale in the Image Display window A click on the Home button resets the display parameters to the default values A click...

Page 460: ...ight from the rear By changing the available parameters one can mix different channels and reveal relationships between information in those channels This view is particularly useful for visualizing t...

Page 461: ...culated by the main processor CPU of the computer Maximum basic or the graphics card Maximum advanced in OpenGL mode Note that this is a rendering If desired 1 1 Maximum Intensity Projections can also...

Page 462: ...e display Using this mode one can render small structures within cells such as vesicles or speckles e g FISH signals as surfaces in one channel of a multichannel image while rendering the surrounding...

Page 463: ...Transparency as well as in the Split mode see below The clipping planes can be accessed via the 3D tab or the Clipping tab On the 3D tab only the most commonly used functionality is available The ful...

Page 464: ...Each plane is placed into the center of the volume and oriented in orthogonal fashion The planes have an opaque surface and colored outlines The clipping functionality of the planes is not activated i...

Page 465: ...s threshold ramp etc applying for Transparent render mode Black pixels are transparent Textured fine Same as above but with 4x times the resolution Tansparent Data which are touched by the plane are d...

Page 466: ...displays or hides the colored outlines The actual color can be chosen from a color chooser for each plane individually Show Plane hides or displays the plane A plane can cut away information whilst b...

Page 467: ...ed the following adjustments can be applied to the datasets All settings can be saved and re applied to other datasets by using the buttons Threshold specifies a lower threshold as a percentage of the...

Page 468: ...slider value range 180 to 180 Channels The settings are entered separately for each channel using sliders or by entering a numerical value in the corresponding input field To select a channel click o...

Page 469: ...meters Click on Apply to start the animation The animation is performed in a separate Image Display window which permits the animation to be saved afterwards 1 Turn around X and Turn around Y mode In...

Page 470: ...included in the list of the Render Series window with a click on the Add button Remove permits the contents of the list to be deleted Insert inserts a position above the highlighted list entry The va...

Page 471: ...ialog contains a toolbar with the available measurement tools The Line tool measures the length along a line in m First click on a starting point and move the mouse to the desired end point while keep...

Page 472: ...tereo views can be chosen from this sub menu Anaglyph is a view in which the data can be examined in 3D using red green glasses The image is built up twice once each for the red and green colors resul...

Page 473: ...range 1 to 100 degree Depending on the object orientation the number of z slices and the chosen render settings this loading process can be visible and may be disturbing especially when rendering a s...

Page 474: ...Using this feature creates render series see section Series with the output size indicated in the two input boxes The maximal resolution is 4096 x 4096 pixels This feature is available in all Render...

Page 475: ...ion of Interest show the histogram values in table form copy table to clipboard or save as text file measure area and mean gray value and standard distribution in an area The settings of the Dimension...

Page 476: ...M OPERATION ZEISS Center Screen Area Image Containers Display and Image Analysis LSM 880 470 000000 2071 464 10 2014 V_01 Fig 666 Image Display Histogram view The Histo button can also be used online...

Page 477: ...of Pixels and Size of Area in an additional table Area measurements of very small areas 10 pixels give only approximate values Show Image tick box Shows the image in the Image Display window with the...

Page 478: ...calization is defined by the presence of two or more different molecules at the same location in a specimen However in the context of digital imaging the term colocalization refers to colors emitted b...

Page 479: ...OPERATION LSM 880 Center Screen Area Image Containers Display and Image Analysis ZEISS 10 2014 V_01 000000 2071 464 473 Fig 669 Image Display Colocalization view Fig 670 Scatter diagram and threshold...

Page 480: ...ly updated when settings are changed Available Controls Drawing tools With the drawing tools the colocalization analysis can be restricted to a region of the image Tools in the Colocalization View Opt...

Page 481: ...f pixels above threshold Value range 0 1 0 no colocalization 1 all pixels colocalize All pixels above background count irrespective of their intensity Weighted colocalization coefficients i i total i...

Page 482: ...in a multi channel image The settings of the Dimensions Display Player and Graphics view options control blocks apply The additional view specific Profile View Option control block is shown in Fig 67...

Page 483: ...880 Center Screen Area Image Containers Display and Image Analysis ZEISS 10 2014 V_01 000000 2071 464 477 Fig 672 Image Display Profile View Line Profile with markers Fig 673 Image Display Profile Vi...

Page 484: ...open arrow Activates the straight profile drawing mode Click into the image and hold the mouse button drag a line in any direction and release the mouse button to end the procedure Open polyline arro...

Page 485: ...ined regions and points are no longer valid Fig 674 Shuttle and Find control tab Options provides settings for the general behaviour of the stage Use DoubleClick in image to move stage Moves the stage...

Page 486: ...the stage to the center of the loaded image ROI POI Moves the stage to the selected ROI POI Show stage position Shows the current stage position as a rectangle in the image Dimension panel Coordinate...

Page 487: ...graphic element To change the name double click in the Name field Then enter the text of your choice Correlating a live image and a snapped image The snapped image and the regions of interest drawn in...

Page 488: ...n of ROI View Option control block is shown in Fig 675 Any changes done with this View Option control block are effective immediately The Image Display in the Mean of ROI View shows 3 panels the inten...

Page 489: ...reen Area Image Containers Display and Image Analysis ZEISS 10 2014 V_01 000000 2071 464 483 Fig 676 Image Display window Mean ROI display for time series in single plane Fig 677 Image Display window...

Page 490: ...use button to end the procedure Recycle bin button All the ROIs to the image are deleted Rectangle and 3D Rectangle button Activates the rectangle drawing mode Click and hold down the mouse button dra...

Page 491: ...elevant ROI in the intensity time diagram Diagram line width pull down sets the line with of the curves in the diagram Diagram graph style pull down choose from a set of graph styles Show Table tick b...

Page 492: ...ay Player and Graphics View Options control blocks apply The additional view specific FRAP View Option control block is shown in Fig 678 Any changes done with this View Option control block are effect...

Page 493: ...y to the graphics view options control block ROIs from both control blocks can be combined Background Region Check box in the list of ROIs Mark the region of interest which represents the mean backgro...

Page 494: ...time identical to the cell which has been bleached to induce FRAP Make sure the whole cell or cell compartment of interest is imaged and therefore illuminated Use the time slider in the Dimensions vi...

Page 495: ...s The rate constant for the exchange of molecules between the bleached region and the surrounding area K per second The part of the immobile fraction of the protein I delta immobile fraction Checking...

Page 496: ...e Containers Display and Image Analysis LSM 880 490 000000 2071 464 10 2014 V_01 Fig 681 Image window displaying the analysis of FRAP data using a mono exponential fit Fig 682 Image window displaying...

Page 497: ...g the table display of the results Note that this modeling is also a very basic approach to your experiment It offers a first hint on the possible presence of two mobile fractions of the labeled prote...

Page 498: ...a view through the oculars without emission filter see Fig 685 The settings of the Graphics View Options control block apply as in 2D view In the Display View Options control block the channel specifi...

Page 499: ...APTER 1 SYSTEM OPERATION LSM 880 Center Screen Area Image Containers Display and Image Analysis ZEISS 10 2014 V_01 000000 2071 464 493 Fig 685 Dimensions view option control block for Lambda Coded Vie...

Page 500: ...hanges done with this View Option control blocks are effective immediately The Image Display in the Unmixing View shows 3 panels the intensity over lambda diagram with the extraction bands 32 channel...

Page 501: ...ER 1 SYSTEM OPERATION LSM 880 Center Screen Area Image Containers Display and Image Analysis ZEISS 10 2014 V_01 000000 2071 464 495 Fig 688 Unmixing Display of a Lambda stack acquired on an LSM 710 LS...

Page 502: ...S Center Screen Area Image Containers Display and Image Analysis LSM 880 496 000000 2071 464 10 2014 V_01 Fig 689 Unmixing Display of a Lambda stack with a spectral dataset acquired on an LSM 700 with...

Page 503: ...n the Image Display window Tables can always be saved or copied to the clipboard by right mouse clicking on the table display Tools for unmixing Overview of spectral data selected that is displayed an...

Page 504: ...ction Processing Linear Unmixing The values of the intensity Lambda diagram are saved to the spectra database in the ZEN DyeDatabase directory Make sure to have displayed only data of single dyes in t...

Page 505: ...e unmix the data set by clicking the button Fig 692 A new and spectrally unmixed multichannel image document is created as many channels as previously defined components Benefit and limitations of the...

Page 506: ...nnel button opens the color selection list and a LUT can be assigned to this channel Add or reduce the number of channels displayed and available for assignment Up to 8 channels labelled after the ass...

Page 507: ...d by right mouse clicking on the table display Fig 693 FRET Display of Image after Sensitized Emission analysis After a Sensitized Emission analysis the table shows the following parameters Fig 693 Re...

Page 508: ...fficiency for each pixel is calculated and averaged for all pixels of the region FRET Eff The averaged intensities of the region are used to calculate an averaged FRET efficiency for this region Delta...

Page 509: ...isplayed in the Threshold tab Regions and the assigned status as Object or Background can be selected or deselected for the individual analysis using the corresponding check box under Enabled To start...

Page 510: ...he defined thresholds will not be displayed in the FRET Image and are not part of the analysis value and area Exclude saturated max intensity pixels Pixels that are saturated in at least one channel w...

Page 511: ...If all further changes shall be effective immediately the On the fly checkbox can be checked Three different methods for FRET analysis are available in the drop down list Fc Youvan FRETN Gordon and N...

Page 512: ...as a fret file by choosing Donor in the drop down list and pressing afterwards the Save button To obtain the Acceptor coefficient values an image of a sample with only acceptor fluorescence is taken...

Page 513: ...f the parameters used for the image analysis The General Fig 698 tab applies to both methods Acceptor Photobleaching and Sensitized Emission If checked the listed options will be taken into account fo...

Page 514: ...st as small as 60 nm Best results are obtained if pixel sizes are 10 fold smaller than the beam waist Another pre requisite is the correct scanning speed This should match the diffusion coefficient of...

Page 515: ...original intensity image and the left one the computed correlation image using a default LUT look up table and a scale bar for the correlation Fig 704 Below the result table is displayed Fig 704 RICS...

Page 516: ...r shaped region can be drawn The correlation will be newly computed taken into account now only the ROI If the Arrow button is active the ROI can be moved around the image by drag and drop If the Arro...

Page 517: ...e pull down menu between Spatial auto correlation S A and Spatial cross correlation S C Fig 707 Channels allow you to select the channel for which a correlation should be performed For a one channel r...

Page 518: ...the Average frames enter box will become available Fig 711 Choose a moving average frame number that would clean up the correlation from slow moving structures by entering a number in the box by usin...

Page 519: ...ctionalities Activate Display under the RICS view The Display options will be displayed in the View control window Fig 713 Fig 713 Display control window In the Representation pull down menu you can c...

Page 520: ...t for different representations different options will become available For the Diagram Table and 2 5D display the Pixel pull down menu becomes available see Fig 717 Here you can choose to display dis...

Page 521: ...y de checking you can enter your own minimum and maximum values into the corresponding Min and Max display boxes by typing or by using the arrows All values between the minimum and maximum will be sca...

Page 522: ...t Pressing the export button below the Hide G 0 box will export the residuals image If Table Image or Diagram was selected from the Representation pull down menu the 2D image will be exported If 2 5 D...

Page 523: ...numbers is lost and diffusion coefficients of the mapped areas You can also display the informative pixels by choosing Inf Pix This will display a map that indicates how many pixels contributed meani...

Page 524: ...ing the slider arrows or typing The default value of a shift size half the region size can be obtained by pressing the 50 region size button In Filter maps different Filter modes can be selected to so...

Page 525: ...k map is filled with white regions Fig 726 The Generate button will only be active if the correlation was fit to a model beforehand to ensure the correct one was selected Only at the end of the map th...

Page 526: ...Fig 727 Fig 727 Fit control window If the Automatic fit check box is selected a fit will performed automatically whenever the correlation input intensity image is changed For example if a region of i...

Page 527: ...ed Terms in the parameter columns can be activated or deactivated by ticking or un ticking the respective check boxes Fig 729 The model pull down menu of the Fit table In the State display area any ab...

Page 528: ...uppressed Pixel dwelling times in s and line times in ms of the scan are displayed in the Pixel times in s and Line times in ms input boxes in the Show all mode They are read from the Meta data of the...

Page 529: ...overwrite the old existing one in the Save Model Warning menu Fig 738 Press Yes to overwrite No to keep the old model Type in a name in the Model name display box and press Ok if you want to save the...

Page 530: ...ta from the image whenever an image is loaded If these Meta data are not available values have to be provided In the Scanning information box the formulas for the Scanning term Gs and the conversion f...

Page 531: ...molecular brightness of the molecules which have to be determined beforehand If unchecked molecular brightness will not be taken into account and the Molecular brightness input box will not be display...

Page 532: ...nus to select between free translational or anomalous translational diffusion for each component Fig 743 Dimension offers selection via pull down menus of one two or three dimensional diffusion Fig 74...

Page 533: ...In the Software the p and s designations are used Polarisation P and anisotropy r are defined as s p s p I I I I P and s p s p I I I I r 2 with 0 r 1 Hence they can be interconverted to each other in...

Page 534: ...ton This opens up the Polarizer selection box Check the polarizer which you want to use with track 1 The options are P for the parallel polarized light transmission and S for vertical polarized light...

Page 535: ...tio Type 1 Set all editable summands to 0 and all factors to 1 see Fig 747 For an inverted microscope use for convenience a glass bottom dish and pipette enough solution in to cover the glass well For...

Page 536: ...in track 1 S in track 2 leaving all other settings equal see Fig 749 Assign track 1 to Ch1 T1 track 2 to Ch1 T2 Alternatively assign to Ch2 T1 and Ch2 T2 or ChS1 T1 and ChS1 T2 whatever physical chann...

Page 537: ...th source 1 S1 being the P track image Ch1 T1 and source 2 S2 the S track image Ch1 T2 with consideration of the G factor see Fig 750 Proceed as follows In the Processing tab select the Image calculat...

Page 538: ...e experiment The user name of the person who acquired the image The pixel scaling in X Y Z The image dimensions pixels in x y z channels bit depth Calibrated image size x y z in m The scan mode e g pl...

Page 539: ...CHAPTER 1 SYSTEM OPERATION LSM 880 Center Screen Area Image Containers Display and Image Analysis ZEISS 10 2014 V_01 000000 2071 464 533 Fig 751 Information View of a processed superresolution image...

Page 540: ...owser Open Images are accessible in the Right Tool Area ZEN File Browser The File Browser allows access to image data of variable image formats and permits displaying them in three views Gallery View...

Page 541: ...cted network drives available on the computer Clicking on a folder opens it and lists the image files therein The ZEN File Browser can be operated in three views Gallery View Table View and Form View...

Page 542: ...Click Split to open the dialog Chose the Size of the dimension you want to split up the image The check box in the Separate Files column activates deactives the split process for the individual dimens...

Page 543: ...2071 464 537 Within the dialog Fig 756 use the editing boxes or the sliders to set the desired values The slider box toggles with the highlighted dimension Click Select file name and save to save the...

Page 544: ...If the CTRL key is pressed all clicked images are selected Opening Images To open an image double click on it For opening multiple images at a time select the desired images and press Load The inform...

Page 545: ...CHAPTER 1 SYSTEM OPERATION LSM 880 Right Tool Area Data Management and Storage ZEISS 10 2014 V_01 000000 2071 464 539 Fig 759 Images with information...

Page 546: ...e of the slider The total number of images in a folder is indicated on the right side of the slider In the Form View of the browser all information available or entered in the Info tab of the image is...

Page 547: ...View is especially useful in case a lot of files are found in one folder The columns displayed in the table are selectable from the Table columns button The options shown in Fig 762 are available The...

Page 548: ...g the following buttons A new acquisition document can be opened using the button The button saves the selected image The button closes the selected image On already saved images the button is closing...

Page 549: ...icking Send to ZEN 2012 blue edition sends the currently active document to ZEN blue edition Regardless of which display type is chosen the individual images contain the following information File nam...

Page 550: ...cilitating the overview if e g many processing function are running at the same time In case long filenames are used the whole section can be dragged to the left and thereby enlarged Fig 768 The stars...

Page 551: ...to be stored in a czi file Proceed as follows to save an acquired or an edited processed image Click on the Save or Save As button in the File menu of the menu bar or click on the icon of the main too...

Page 552: ...be exported to Fig 771 Select the data type which the image is to be exported under Data Fig 772 Chose a compression level For some file formats lossless compression or various other compression leve...

Page 553: ...itude 198 214 Analog display 122 Animation 428 Antibunching 215 Anti bunching terms 201 AOTF dampening 417 Aperture control 89 Application bar 32 Arrow down button 47 Auto save 418 Automatic Component...

Page 554: ...images 546 Expose mode 46 Exposure 73 F Fast iterative 365 FCS correlation diagram 268 FCS diagram 260 FCS measurements 260 FCS options 413 FCS tab 175 File 34 Filter 305 Filter cube selection 86 Fil...

Page 555: ...view processing 364 Online multiview processing 381 Pre Registration 372 Registration 368 372 Registration options 368 Use registration from file 375 Line 106 Linear unmixing 308 Live 73 Locate tab 51...

Page 556: ...annel alignment 354 Color coded projection 301 Copy 382 Correlation 319 Experimental PSF 341 Filter 305 HDR imaging 321 ICS 384 Image calculator 302 Ion concentration 313 Lightsheet processing 361 Lin...

Page 557: ...red illumination 347 TIRF Filtered 352 Subset 382 383 System options 409 FCS 413 Hardware 411 Image display 412 Load configurations 410 Reuse 410 System startup 18 T Text view 47 Textual view button 4...

Page 558: ...CHAPTER 1 SYSTEM OPERATION ZEISS Index LSM 880 552 000000 2071 464 10 2014 V_01 Zoom function 275 284 Z Piezo 146 Z Stack 141 Z Stack settings for Airyscan 149...

Page 559: ...ionFingerprinting 11 2 6 ConcatenateImages 14 3 Visual Macro Editor 15 3 1 Open Close the Visual Macro Editor Window 15 3 2 Function Description 16 3 3 Editing a Macro 23 4 VBA Programming for LSM 26...

Page 560: ...SUAL BASIC ZEISS Contents LSM 880 2 000000 2071 464 10 2014 V_01 4 8 Example Macros 33 4 8 1 Access to images 33 4 8 2 Backup recording settings 33 4 8 3 Event handling 33 4 8 4 Export Save and Load 3...

Page 561: ...ssign macros to list Visual Macro Editor edit automatic workflows with pre defined modules Selecting Macro opens a dialogue for editing and recording Macros based on VBA programming in the Macro Contr...

Page 562: ...acro Function This function allows you to manage project data Macros can be recorded stored performed edited and if required deleted Press the Edit Macro tab to switch to the Macro and Recording panel...

Page 563: ...be opened see Fig 5 Select the relevant project file data extension lvb from the Macros list box Click on the Open button The project file will be opened and the macros contained in it are displayed i...

Page 564: ...button A new Image Display window will be opened Click on the LSM Snap action button A scan will be performed Then click on the Stop button to end the recording Cancel enables you to cancel recording...

Page 565: ...nel see Fig 8 Define Buttons panel Proceed as follows to link a macro to the Macro list Select the button number corresponding to the position in the list from the Button selection box Enter the butto...

Page 566: ...lines Stops running the command lines Interrupts processing of the command lines pause Sets a breakpoint in the line with the text cursor Processes a command line and steps into subprocedures Processe...

Page 567: ...as separate pdf file GDDReset lvb Enables or disables the use of the factory set GDD values Resets the GDD settings to factory values if other values have been stored over time LsmHWAdmin60 lvb also B...

Page 568: ...lled in ZEN Macros Self generated Macros can be saved in any folder In case of a new installation old macros will be stored in ZEN Macros BackupMacros or ZEN Backup to avoid problems with identical na...

Page 569: ...otherwise the scan will not start and no laser is available When ZEN is started one channel is always active per default If the system was in use check in the Imaging Setup tool if a channel is active...

Page 570: ...6 Activate the Auto option Auto is chosen when starting with the highest wavelength available 7 By clicking Add and 10 Steps the AOM value will be added to the calibration list and the laser is tuned...

Page 571: ...the AOM and detector gain to obtain a good image at the chosen wavelength Switch to Excitation Lambda Stack 2 Load the calibration file required for the used main dichroic and objective 3 Set the wave...

Page 572: ...of one Block only In this case the time stamps of the image are consecutive relating to the image acquisition time of the first image of the Block Use Raw image date Mode and Size Union for concatena...

Page 573: ...which allows the user to program a variety of scanning procedures image calculations or image processing routines as well as the combination of these functions 3 1 Open Close the Visual Macro Editor W...

Page 574: ...d variables is displayed New ones can be added by typing in name and value in the next free line Variables are assigned to macros For each new macro a new list of variables has to be generated The edi...

Page 575: ...or Click onto an output arrow with the left mouse button hold down the mouse button and drag the mouse cursor away A connecting line appears Drag the line to the input arrow of another action block On...

Page 576: ...or example the zoom at a certain position within the program flow as for each block the check boxes can be set This block performs the scanning of the sample according to the settings chosen with Beam...

Page 577: ...e focal plane for imaging Assign position to Chose a variable to which the new position of the focus plane set by the auto focus function e g coverslip reflex should be assigned Assign offset to Chose...

Page 578: ...ped into the value line All predefined variables can also be used for calculations By passing this block the variable will be transiently changed and the new value can for example be used to assign di...

Page 579: ...ues of the listed properties Using this block enables to copy one image into another with the coordinates and settings put into the properties list The destination image is connected to the left input...

Page 580: ...loaded images Intensity range Defines the intensity range which is applied for mask definition Lower threshold Defines lower intensity threshold which is applied for mask definition This threshold can...

Page 581: ...puts and outputs for each flow is ten Using this block allows integrating macros or macro procedures programmed with visual basic into a VME macro The number of data flow inputs and outputs can be set...

Page 582: ...ck The last one used will be taken To update this open the desired database and use the Read back function Type in a name for the images with or without quotation marks All images will get the same na...

Page 583: ...iable Index is chosen and the value assigned is Index 1 Such the variable will get the transient value 2 for the next cycle the value 3 for the over next cycle and so on The number of repeats can be d...

Page 584: ...API offers a low level hardware near access to the ZEN system It is the older of the two APIs and has been used with ZEN macros for many years Because of this some of its functionality has changed it...

Page 585: ...t use the object browser from the VBA editor To explore an object from your VBA code position the curser in the object and press F2 For the Lsm5Vba API many classes and methods offer a short help stri...

Page 586: ...sm5Vba Application ZEN actxGetRunningServer Zeiss Micro AIM ApplicationInterface ApplicationInterface To start a scan two commands are now available ZENVBA Lsm5 StartScan ZEN GUI Acquisition Snap Exec...

Page 587: ...lifiers CpCollimators CpFilterSets CpFocus CpIntegrators CpLamps CpLaserLines CpLasers CpObjectiveRevolvers CpPinholes CpPmts CpScanControl CpServos CpShutters CpStages CpTriggers DsGuidedModeDatabase...

Page 588: ...he ZEN VBA GUI interface is to make ZEN commands available in the same way as they are in the GUI The image shows how this principle works To set the acquisition frame size one has to 1 Select the Acq...

Page 589: ...or write the corresponding value For numeric values the command also offers read only Minimum and Maximum properties A command corresponding to a range of options i e a combo bvgbox or drop down box...

Page 590: ...obtained directly from here 4 7 5 Technical note The ZEN VBA GUI interface is a new component for the 2011 release of ZEN black edition only It is not backwards compatible with earlier releases of ZE...

Page 591: ...xamplesMacros access_to_images lvb 4 8 2 Backup recording settings This macro demonstrates how to backup the acquisition settings and then later change the settings back to the original ones C ZEN Mac...

Page 592: ...example macros for the VBA GUI API because these can easily be generated by the macro recorder functionality Activate macro recording and the execute the actions that you want to automate Then edit t...

Page 593: ...2014 V_01 000000 2071 464 1 CHAPTER 3 ADDITIONAL SOFTWARE TOOLS CONTENTS Page 1 Stand Select 2 2 SystemMaintenance Tool 3 2 1 Calibration Objective 3 2 2 Starting the Maintenance Tool 4 2 3 General O...

Page 594: ...he screen Click on the name of the database file extension mdb and then the Open button The Open window is closed and the name of the new database appears in the Database box Click on the Permanent bu...

Page 595: ...not suitable for multiphoton excitation there is a risk of bubble formation in the substrate Using the HAL the IR protection filter must be used and the intensity of the HAL should only briefly be set...

Page 596: ...eds to be entered as APO Calibration LSM For LSM systems with a 405 nm laser an approximate collimator position should be saved for the calibration objective The focus plane needs to be approximately...

Page 597: ...Sharpness Test Scan Field Test Scanner Calibration Pinhole to MBS Calibration In case a task requires any manual adjustments the Maintenance Tool guides users to all adjustment steps After the comple...

Page 598: ...3 ADDITIONAL SOFTWARE TOOLS ZEISS SystemMaintenance Tool LSM 880 6 000000 2071 464 10 2014 V_01 Fig 9 Maintenance Tool main window Fig 10 The Maintenance Tool guides users through required adjustment...

Page 599: ...atabase before you start to configure it Do not change any main settings or your system will not work anymore The following procedure describes how to define a new filter for external detectors like N...

Page 600: ...drop down list next to Filterwheel choose the device you want to define a new filter for This is either Emissionfilters for DC Out detectors BiG 2 on DCout port Emissionfilters for reflection detector...

Page 601: ...rring to the detection channel it is effective for Fig 12 Clicking this button opens the Change Filter window with a list of favourite potential and user defined filters Fig 14 To define a new filter...

Page 602: ...ation to the system so it can use the information for image acquisition and processing The form on how to fill in the information is displayed as example in the lower part of the window when editing a...

Page 603: ...Excitation 8 3 4 Acquisition Conditions for RICS 8 4 Recommendations for Excitation Laser Lines and Emission Filters of Dyes 9 5 How to Access Data Acquired with Airyscan Using Other Software 10 6 Pie...

Page 604: ...ZEN Module FRAP Efficiency Analysis Software ZEN Module FRET Software ZEN Module Visual Macro Editor VME Software ZEN Module Image Correlation Spectroscopy RICS If your configuration does not include...

Page 605: ...esigner tool If your configuration does not include the ZEN Module 3D VisArt the following functions are only available in a basic stripped down version 3D view type in the Image Display document If y...

Page 606: ...vice or the Z Piezo stage the following functions are not available Use Piezo in the Z Stack tool in the left tool area If your configuration does not include an X Y scanning stage the following funct...

Page 607: ...3 1 Optical Elements in the Configuration Control Window All wavelength values are given in Nanometer nm 3 1 1 Main Dichroic Beam Splitter MBS A MBS XXX YYY deflects the indicated laser lines onto the...

Page 608: ...nt of ChS1 or Ch2 The active detection bands may not include any of the active laser lines however this will not interfere with imaging Additional hints Do not forget to turn on the corresponding lase...

Page 609: ...464 7 Add a new track by clicking the button next to Tracks in the Imaging Setup tool Create a second track for AF 568 By selecting the dyes from the pull down menu you can see the emission spectra to...

Page 610: ...en Frame mode is used There is a fast Frame mode which requires identical settings of these parameters 3 3 Beam Path Configuration for Multi Photon Excitation The beam path configuration for multi pho...

Page 611: ...Alexa Fluor 488 488 505 max at 520 Calcium Green 488 505 max at 531 Cy2 488 505 max at 508 DiO DiOC18 3 488 505 max at 508 Fluo 3 488 505 max at 520 Fluorescein FITC 488 505 max at 520 Cy3 514 532 53...

Page 612: ...e in dimension H If you can read CZI files you can directly access the Airyscan raw data channels They have 32 entries in the H dimension The documentation of the CZI file format can be requested via...

Page 613: ...e of a Z stack The available travel range of a Z Piezo device is much smaller compared to the Z drive of the microscope A Piezo device allows performing smaller steps in Z with a higher absolute preci...

Page 614: ...s M27 Installation Screw in your microscope objective into Piezo Objective Focusing Device see Fig 3 1 Screw the thread ring into your microscope see Fig 3 2 Easy clamp the Piezo Objective Focusing De...

Page 615: ...signals The second interface is accessible via the software user interface in the tools Time Series and Bleaching and can be used to accept or provide signals for the following actions Change interval...

Page 616: ...of the TTL pulse therefore is 1 2 x pixel rate 15 point high density D Type plug Plug A Pin Signal name Description Software interface 1 SyncOUT0 synch output Synchronous output Trigger 1 out 2 SyncOU...

Page 617: ...switches on the UserPortAdapter inside the RT PC must switch thrown towards enclosure default position Timing of the synchronization signals Stack out Line out and Frame out These signals are directly...

Page 618: ...o 3 3 V Trigger pulse description Signal output Low level 0 4 V high level 2 4 V I 10 mA Slew rate 10 ns V Reaction time trigger OUT 8 msec typical Signal input Low level 0 5 0 8 V high level 2 0 5 5...

Page 619: ...the user interface on the realtime electronics Fig 4 Panel of the User Interface box Trigger pulse description The pulses are identical to the pulses described for the user interface on the realtime e...

Page 620: ...veling in X in the following way a Enter in the Stage tool in the Step m input box 1000 Press the upper or lower pointing arrow of the X input box whatever is required to step 1000 m to the right X di...

Page 621: ...positioned in the center d Use the front right screw Fig 5 sy of the sample holder to readjust the focus Fig 6 E Turn clockwise if the focus position was positive counter clockwise if it was negative...

Page 622: ...ing planes are wavy or you get reflection from different planes Fig 8 C 4 Move in 10x objective and focus the already visible ring with the z drive of the stand The ring appears now well focused as lo...

Page 623: ...e holder schematic view Fig 10 Focus control with focus drive Course of reflection on tilted A and even B coverslip when moving focus upwards left to right 0 Adjust the edges of the clefts viewed from...

Page 624: ...d Any tilt will appear as a brightness gradient or even a bright zone or line that wanders through the slices Fig 10 6 Adjust the adjustment screws of the sample holder Turn screw sx for the tilt alon...

Page 625: ...with about 80 slices and an interval of 0 2 m Activate z Piezo Click on the Center button and the start the Z Stack with a long time series Use Corr X Corr Y and Corr Z to remove jitter as much as pos...

Page 626: ...ur times No cooling liquid is in the system Fill the system with liquid A corresponding acoustic signal is provided along with the optic signal It is recommended to exchange the liquid every two years...

Page 627: ...els tool 4 1 3 Motorized Periscope 5 1 3 1 Instructions for beam adjustment using the motorized periscope 6 1 4 Pinhole and Collimator Settings 11 2 Non Descanned Detection NDD 12 2 1 Thickness of opt...

Page 628: ...ake up to one hour Specific for the operation of Chameleon lasers Coherent for further details please refer to the operators manual of the laser The key on the front panel of the laser power supply is...

Page 629: ...pumping the OPO If the laser is equipped with a pre compensation unit prechirp unit like the versions Chameleon Vision or Mai Tai DeepSee a slider and edit box show up in the Laser Properties panel al...

Page 630: ...led via a manual neutral density filter Changing the intensity is possible via a slider in the Imaging Setup tool or the Channels tool Note With the ND filter as attenuator it is not possible to perfo...

Page 631: ...e Corrections should only be done in small steps and should only be necessary for controlling the overlay of the two beams if beams are to be used for the excitation of the same area simultaneously or...

Page 632: ...allow optimizing the position of the acquired image and the optimization of the illumination profile of this image It might be necessary to optimize the image position and or illumination due to devia...

Page 633: ...wavelength range NLO laser short wavelength range Input and Output correspond to the position of the mirror inside the periscope as described above Each axis of each mirror can be controlled independe...

Page 634: ...lateau is adjusted by clicking on the buttons representing Axis 2 1 3 1 2 Correction of image position If a sample is excited using two NLO lasers or an NLO laser and OPO the corresponding two images...

Page 635: ...e right fine movement button of Beam1 Output Axis2 repeatedly until an image shift and illumination change is visible image gets darker 2 Now click on the right fine movement button of Beam1 Input Axi...

Page 636: ...repeatedly until an image shift and illumination change is visible image gets darker 2 Now press the left fine movement button of Beam1 Input Axis1 repeatedly until the image is bright again The imag...

Page 637: ...lect the NLO collimator in the Collimator panel It is best to use a fine structured object like beads which are excited with visible lasers and the NLO laser Focus on the bead and acquire an image wit...

Page 638: ...oosing the Non Descanned tab next to the LSM tab This tab is only available when NDD detectors are connected Click on the Non Descanned tab to change to Non Descanned Detection After checking a box ne...

Page 639: ...y the blind plate otherwise the system is not safe and does not work Make sure not to use two modules of the same type on one LSM system This leads to errors in the system and one of the modules will...

Page 640: ...ected via sub LVDS to the realtime electronics as listed above The NDD channels 3 and 4 only have to be connected to the 2 channel NDD module but don t need a connection to the realtime electronics Fi...

Page 641: ...on NDD ZEISS 10 2014 V_01 000000 2071 464 15 In addition the power connections have to be made in the following way to the plugs on the back of the electronics rack Fig 9 Power connections of NDD dete...

Page 642: ...stics may lead to inferior image quality All secondary dichroic filters for the NDDs Push Click filter cubes are long pass filters They split the emitted fluorescence according to the indicated wavele...

Page 643: ...bed in Fig 12 As the dichroic filters are also long pass filters channel 1 always needs to see the red part of the spectrum followed by the blue part of the spectrum within the required detection rang...

Page 644: ...rs in the filter cube for the green channel in front of channel three is interchanged with the emission filter of the filter cube for the orange signal which sits in front of channel one The cube with...

Page 645: ...Reflected Light NDD Port BiG NDD as channel 1 2 NDD module type A 2 channels for upright microscopes BiG NDD module as channel 3 4 For Axio Examiner BiG 2 with GaAsP at Transmitted Light NDD Port BiG...

Page 646: ...s channel 3 4 BiG 2 with GaAsP at Transmitted Light NDD Port External detection module BiG DC transmitted light BiG NDD as channel 1 2 no additional NDD modules can be attached Once the NDD modules ar...

Page 647: ...e the tab Objectives In the top most drop down list choose the 1 position Objective nosepiece manual Fig 15 Fig 15 Configuration tool with Microscope and Objectives tab active for choosing the objecti...

Page 648: ...d remove the module How to install a module Insert the filter module Fig 18 4 with the aid of its mounting elements on its left and right 318HFig 18 3 into the lower spring clips 319HFig 18 1 on the f...

Page 649: ...graph comprehensively deals with the quality parameters of resolution depth discrimination noise and digitization as well as their mutual interaction The set of equations presented allows in depth the...

Page 650: ......

Page 651: ...Confocal Laser Scanning Microscopy Stefan Wilhelm Carl Zeiss Microscopy GmbH Carl Zeiss Promenade 10 07745 Jena Germany...

Page 652: ...7 Signal Processing Part 2 limits the idealized view looking at the digitizing process and the noise introduced by the light as well as by the optoelectronic components of the system Sampling and Digi...

Page 653: ...confocal Laser Scanning Microscope LSM has become widely established as a research instrument The present brochure aims at giving a scientifically sound survey of the special nature of image formation...

Page 654: ...applications will be given priority treatment throughout Image generation The complete generation of the two dimensional object information from the focal plane object plane of a confo cal LSM essent...

Page 655: ...ich illuminates only a point of the object at a time the point illuminated and the point observed i e image and object points are situated in conjugate planes i e they are focused onto each other The...

Page 656: ...a thick biological specimen will only be in focus if its Z dimension is not greater than the wave optical depth of focus specified for the respective objective lens Unless this condition is satisfied...

Page 657: ...n cytoplasm and nuclei of acinar cells with ethidium homodimer 1 red Fig 5 Gallery of a time series experiment with Kaede transfected cells By repeated activation of the Kaede marker green to red colo...

Page 658: ...3D PSF In the ideal diffraction limited case no optical aberrations and homogeneous illumination at all lens cross sections see Part 3 Pupil Illumination the 3D PSF is of comet like rotationally symme...

Page 659: ...n n2 NA2 2 n PH NA 2 2 0 88 exc n n2 NA2 5 1 77 n exc NA2 FWHMill lateral 0 51 exc NA seite 12 PSFtot x y z PSFill x y z 2 em exc 2 exc 2 em 5 2 0 64 1 28 n 1 X Z X Y PSFdet is also influenced by all...

Page 660: ...epth discrimination With a pinhole diameter greater than 1 AU AU Airy unit see Details Optical Coordinates the depth discriminating properties under consideration are essentially based on the law of g...

Page 661: ...o the conventional fluorescence microscope confocal fluorescence with large pinhole diameters leads to a gain in resolution by the factor em exc via the stokes shift If NA 0 5 equation 2 can be approx...

Page 662: ...e sentation The center has a normalized intensity of 1 The real relationships result by rotation of the section about the vertical Z axis Symmetry exists relative to the focal plane as well as to the...

Page 663: ...ase of geometric optical confocality there is a linear relationship between depth discrimination and pinhole diameter As the pinhole diameter is constricted depth discrimination improves i e the optic...

Page 664: ...z PSFill x y z 2 em exc 2 exc 2 em 5 2 FWHMtot axial 0 64 n n2 NA2 5 1 28 n NA2 FWHMtot lateral 0 37 NA 5 seite 9 FWHMill axial seite 11 FWHMdet axial 0 88 em n n2 NA2 2 n PH NA 2 2 0 88 exc n n2 NA2...

Page 665: ...le though to transfer the equations derived for PH 0 to the pinhole diameter range up to 1 AU to a good approximation The factors applicable to particular pinhole diameters can be taken from figure 10...

Page 666: ...2 5 3 0 3 5 4 0 4 5 5 0 Pinhole diameter AU FWHM m Depth resolution PH 0 n 1 52 496 nm 1000 920 840 760 680 600 520 440 360 280 200 600 560 520 480 440 400 360 320 280 240 200 1 1 1 1 2 1 3 1 4 Numeri...

Page 667: ...oscopy 1 AU PH Confocal microscopy PH 0 25 AU Optical Image Formation PART 1 seite 15 0 88 em n n2 NA2 2 n PH NA 2 2 0 64 n n2 NA2 0 64 n n2 NA2 0 88 exc n n2 NA2 n em NA2 1 77 n em NA2 0 51 em NA 1 7...

Page 668: ...mage formation is extremely small This is due to the efficiency of the system as a whole and the influencing factors involved such as quantum yield bleaching and saturation of fluorochromes the transm...

Page 669: ...see figure 12 Integration The signal detection time has the same order of magnitude as the pixel time Integration is equivalent to an averaging of intensities over a certain percentage of the pixel ti...

Page 670: ...on spacing of sampling points 0 25 AU Fig 14 Oversampling correct sampling and undersampling of a continous signal Nyquist theorem It is known from Part 1 that the information content of the signal is...

Page 671: ...t also the resolution proper ties of the system With the more recent LSM systems from Carl Zeiss the number of sampling points can also be influenced by an increase in the number of pixels per scan li...

Page 672: ...rces of noise are of a sta tistical nature Periodic noise rarely occurs and if it does it tends to correlate with defective devices or mechanical vibration in the setup therefore it has been left out...

Page 673: ...focal microscope PSF with the influences of shot noise of the detected light and the scanning and digitization of the object The essential criterion is the discernability of object details 23 Figure 1...

Page 674: ...ient between suc cessful experiments resolved and the total number of experiments A resolution probability of 70 means that 7 out of 10 experiments lead to resolved structures A probability 90 is impe...

Page 675: ...ging method is the lower laser load on the specimen as the exposure time per 25 pixel remains constant Photon statistics are improved by the addition of photons from several scanning runs SNR n N N co...

Page 676: ...tween resolution and noise in a confocal LSM is solved by way of the concept of resolution probability i e the unrestricted validity of the findings described in Part 1 is always de pendent on a suffi...

Page 677: ...aximum of an intensity distribution e g optical slice n Refractive index of an immersion liquid NA Numerical aperture of a microscope objective PH Pinhole diaphragm of variable size arranged in the be...

Page 678: ......

Page 679: ...to Laser Scanning Microscopy the following pages treat several aspects of particular importance for practical work with a Laser Scanning Microscope Pupil Illumination Optical Coordinates Fluorescence...

Page 680: ...ty distribution in the objec tives pupil plane backfocal plane Hence it follows that a homogeneous distribution in the pupil plane results in a focal distribution following the Airy function also know...

Page 681: ...confocal microscope it suggests itself to express all lateral sizes as multiples of the Airy disk diameter Accordingly the Airy unit AU is defined as The AU is primarily used for normalizing the pinh...

Page 682: ...most important contrasting methods in biological confocal microscopy Cellular structures can be specifically labeled with dyes fluorescent dyes fluorochromes or fluorophores in vari ous ways Let the m...

Page 683: ...except within certain limits As soon as a certain molecule packing density is exceeded other effects e g quenching drastically reduce the quantum yield despite higher dye concentration Another problem...

Page 684: ...re a great number of photons N 10000 are detected Shot noise Poisson noise Shot noise is caused by the quantum nature of light Pho tons with the energy h hit the sensor at randomly dis tributed time i...

Page 685: ...ss and contrast transfer in coherent confocal microscopy Journal of Microscopy Vol 172 pp 31 39 10 1993 11 Pawley J Handbook of Biological Confocal Microscopy Springer 3rd Edition 2006 12 Stelzer E H...

Page 686: ...10 ISBN 978 3 940885 02 9 We gratefully acknowledge the assistance of Bernhard Gr bler Hartmut Heinz and all other staff members who contributed to this brochure Carl Zeiss Microscopy GmbH 07740 Jena...

Page 687: ......

Page 688: ......

Page 689: ...the Deutsches Museum in Munich 1988 The LSM 10 a confocal system with two fluorescence channels 1991 The LSM 310 combines confocal laser scanning microscopy with state of the art computer technology...

Page 690: ...graph comprehensively deals with the quality parameters of resolution depth discrimination noise and digitization as well as their mutual interaction The set of equations presented allows in depth the...

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