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Zeiss ELYRA 7, Operating Manual


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March 2019 

ZEN 3.0 SR (black edition)

 

Summary of Contents for ELYRA 7

Page 1: ...ELYRA 7 Operating Manual March 2019 ZEN 3 0 SR black edition...

Page 2: ...pecifications 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: ...ll 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 the lam...

Page 4: ...typical application examples including the WINDOWS graphic user environment 2 Macros and Visual Basic This section contains a description of the use of additional functions e g maintenance macros 3 A...

Page 5: ...Schematic 13 2 3 Performance and Features of ELYRA 15 2 4 Microscope Equipment of ELYRA Systems 16 2 5 Computer Hardware and Software 18 3 STARTUP AND SHUTDOWN OF THE SYSTEM 19 3 1 Startup of the Sys...

Page 6: ...Beam Path Configuration 71 5 2 5 3 Camera Selection 73 5 2 5 4 Tube Lens Selection 75 5 2 5 5 Filter Cube Selection 75 5 2 5 6 Objective Selection 75 5 2 5 7 HAL Control 76 5 2 5 8 Epi Illumination Co...

Page 7: ...ive when Moving the Stage 146 5 2 21 4 Scan Overview Image 146 5 2 22 Multidimensional Acquisition Information on Experiment 147 5 2 23 Multidimensional Acquisition Streaming and Auto Save 147 5 2 23...

Page 8: ...emove Structure 254 5 3 23 3 ICS Correlation 255 5 3 23 4 Map Filter 256 5 3 23 5 NandB Analysis 257 5 3 24 Processing Adjust 260 5 3 24 1 Burn in Brightness and Contrast 260 5 3 24 2 Interpolate Brig...

Page 9: ...rojection 308 6 8 2 Transparency Render Mode 311 6 8 3 Maximum Mode 312 6 8 4 Surface Render Mode 312 6 8 5 Mixed Render Mode 313 6 8 6 Clipping Planes 314 6 8 7 Flying Mode 318 6 8 8 3D Rendering Set...

Page 10: ...Drift Correction 378 6 18 5 SMLM Grouping View Control Tab 383 6 18 6 SMLM Statistics View Control Tab 385 6 18 7 SMLM Filter View Control Tab 392 6 18 8 SMLM Render View Control Tab 395 6 18 8 1 Ren...

Page 11: ...the images It is a special user interface desktop based on the network capable graphic 64 bit Microsoft WINDOWS 7 10 operating system Portions Copyright 2007 Microsoft Corporation All rights reserved...

Page 12: ...ff 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 Cl...

Page 13: ...enhance resolution the PSF has to be narrowed which is synonymous to the OTF to be expanded This exactly is accomplished by structured illumination microscopy SIM and photo activated localization mic...

Page 14: ...the 0 th order non diffracted beam the 1 st order created by interferenes of the 1 st order diffraction beams with the 0th order non diffracted beam frequencies shifted by half the modulation frequenc...

Page 15: ...photons Fig 5 To image molecules one at a time in a PSF requires certain properties of the fluorescence dye It has to exist in a bright and dark state which can be converted between each other Most of...

Page 16: ...them in a new coordinate system and deactivate them quickly preferentially after 1 to 3 frames and repeat the process until statistically all molecules have been activated So a SMLM experiment easily...

Page 17: ...k focal plane of the objective to obtain widefield illumination in the specimen For TIRF mode dependent on the angle of the TIRF mirror either Epi HILO or TIRF illumination is achieved For SIM mode on...

Page 18: ...ort SPl of the stand The Duolink will always be mounted if present on the right side port SPr For ELYRA 7 type L systems an Andor iXon 897 Ultra EMCCD camera is mounted on the right side port SPr For...

Page 19: ...ers AOTF adjust the necessary brightness for all 4 laser lines within microseconds The 405 laser is in addition equipped with neutral density filters that allow tuning the laser power further down The...

Page 20: ...orescence filter sets you need to employ a further reflector turret When changing the reflector turret position you must make sure that the turret will click into position since otherwise the image ar...

Page 21: ...conventional analyzer slider in the stand must not be located in the beam path because the laser light is already polarized The focusing screen for conventional transmitted light is located in a supp...

Page 22: ...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 ELYRA 7...

Page 23: ...ve access to the software you have to switch the system on and start the computer with the following Booting procedure Switch on the system via the Main switch of the ELYRA 7 system Switch on all thre...

Page 24: ...symbol the context sensitive Help menu will appear Fig 10 Close the menu with the Windows commands if you do not want to search the help database by pressing the x cross symbol Pressing the x cross sy...

Page 25: ...ware in this mode Image Processing for data analysis only This mode ignores the hardware and activates only data handling and image processing functionality to analyze stored images Note that only tho...

Page 26: ...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 27: ...ystem Options dialog window activate Laser off on Exit in the Shutdown tab section Options tool group System Options 3 2 1 Exiting ZEN Software The software can be shut down from File menu of the Main...

Page 28: ...ed changes to exit the ZEN software 3 2 2 Switching System Power Off After having exited the ZEN software you may turn off the whole system with the following Shutdown procedure Make sure you have sav...

Page 29: ...yout ZEISS 03 2019 V_02 000000 2262 999 25 4 Introduction to the Software Application Layout 4 1 Overview on the Screen Layout Fig 17 ZEN Main Application window after Startup with empty image contain...

Page 30: ...rkflow 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...

Page 31: ...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 32: ...ar to where you want it Fig 20 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 33: ...Tool group Tool closed or tool window opened Panel e g Averaging 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 cl...

Page 34: ...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 35: ...enu bar contains 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 21...

Page 36: ...CHAPTER 1 SYSTEM OPERATION ZEISS Introduction to the Software Application Layout ELYRA 7 32 000000 2262 999 03 2019 V_02 Menu overview...

Page 37: ...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 38: ...lance of a connected camera Click on Camera in the Maintain list The Camera Color Adjustment window appears on the screen see Fig 25 Clicking the Pic button allows to set the white balance using the m...

Page 39: ...as the ELYRA system Enable the Remote Access and define a user name and password for the access which then must be used to login form the iPad Download the ZEISS Remote App from the App Store and inst...

Page 40: ...00 2262 999 03 2019 V_02 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 27 About window The panel hosts important inf...

Page 41: ...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 42: ...2262 999 03 2019 V_02 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 43: ...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 31 Progress bar System information Shows information of the current...

Page 44: ...Acquisition Main tool to control the image acquisition process Tool groups Groups special tools e g Setup Manager or Acquisition Parameter Setup Manager Those tools are necessary if the system should...

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Page 49: ...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 50: ...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 51: ...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 52: ...ion Layout ELYRA 7 48 000000 2262 999 03 2019 V_02 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 53: ...rganized in the four Main tool tabs Fig 42 Locate Visual and camera inspection in transmitted and epifluorescence light Acquisition Imaging with ELYRA Processing Processing superresolution and LSM ima...

Page 54: ...utton All saved ocular configurations will be listed in the Load Configuration panel Fig 44 Please note that the selected objective lens and tube lens as well as the position of the 3D SMLM slider wil...

Page 55: ...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 56: ...ntains the following functions HAL On Off button for transmitted light If the button is pressed the control panel appears Fig 50 If you press the On button it will be highlighted and the Halogen lamp...

Page 57: ...urret 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 53 By pres...

Page 58: ...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 59: ...ontrolled via SW The light path will be highlighted in the diagram Press the On button again to shut down the light The button will go to the Off position and the shutter closes automatically Excite R...

Page 60: ...s 1x will be chosen automatically when the Locate tab of the main tool bar is pressed For reflected light observation Epi fluorescence Turn on the power supply switch of the epi illumination light sou...

Page 61: ...in the Transmitted Light panel and set the transmitted light intensity via the slider or click on 3200 K Click on the Condenser button and set the aperture via the slider in the Condenser panel Set th...

Page 62: ...s 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 concentration via the input boxes...

Page 63: ...lumination microscopy SIM It depends on the hardware configuration which of these functions are available All widefield techniques use cameras For excitation all imaging modes use lasers with the exce...

Page 64: ...n 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 parameter typ...

Page 65: ...d as a setting Time series Cycles and Interval Z Stack Range Slices Interval Use of Piezo Bleaching Start and Repeat bleach settings Iterations Stop at specific intensity Different scan speed Trigger...

Page 66: ...AF Find Focus button 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 l...

Page 67: ...es after its activation Fig 72 Fig 72 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 68: ...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 69: ...ons The progress of the experiment in regard to which image the system currently acquires will be shown updated to the right of the functions if available Fig 77 Fig 77 Multidimensional input field di...

Page 70: ...to manually switch off still active lasers before the software closes In case the status On is not changed lasers are set into standby mode if applicable Lasers without power status are switched off...

Page 71: ...or detection Laser WF Laser widefield imaging including epi fluorescence Epi Highly Inclined and Laminated Optical sheet HILO and Total Internal Reflection Fluorescence TIRF modes using a laser for wi...

Page 72: ...evices like filters is possible All mechanical settings for the first track will be applied to the second track as well and an Info message Several settings will appear Fig 83 Press Yes to continue wi...

Page 73: ...rack Note that the EPI and TIRF button usage pressed or depressed will not be updated but the values will E g if in every track in TIRF Angle the button EPI was pressed and now in one track the TIRF b...

Page 74: ...tions will be available for the SMLM image including Drift correction and Grouping It should be noted that a continuous drift will be expressed as jumps at the transition points between the consecutiv...

Page 75: ...ELYRA 7 type the presence of Duolink and Imaging mode selected what components are accessible Some components are common to all imaging modes whereas others will be specific Fig 88 Beam path configur...

Page 76: ...rack has been selected and would not perform an image acquisition To activate the camera again you have to switch between different imaging modes E g if you have inactivated the camera in the Laser WF...

Page 77: ...respective TV1 2 EM CCD Andor SMLM sCMOS pco SIM button which becomes highlighted when selected Always one camera will be active Whenever one camera is active the other will be inactive as they canno...

Page 78: ...hannel from a look up table that becomes available when the TV icon is pressed Fig 92 More than the displayed colors can be obtained by using the scroll bar in the pop up menu The position of the side...

Page 79: ...ination is selected The other position position 2 can be equipped with a cube that is suitable for widefield excitation and observation through the eyepieces Positions 3 6 hold calibrated filter cubes...

Page 80: ...only available for WF imaging mode for reflected light of the lamp is pressed the control panel for the light settings is displayed for software controlled lamps only Fig 97 If you press the On butto...

Page 81: ...IRF The selected mode will be displayed above the icon Note that the optics for TIRF_uHP is not aligned and hence the FOV can be offset from a center position and the evenness of the field might be le...

Page 82: ...HILO and TIRF mode as options TIRF angle setting is specific for the Laser WF imaging mode Press the TIRF angle button to open the TIRF angle settings panel Fig 103 You can switch between EPI HILO an...

Page 83: ...solution you might dilute tap water with distilled water until you have a good ratio between settled and floating beads 3 Mount the glass bottom dish onto the microscope using an appropriate TIRF obj...

Page 84: ...IRF angle button to store the setting Whenever the same laser line is chosen the TIRF angle will be set to the stored position 10 You might repeat with other laser lines albeit the beads of the kit ha...

Page 85: ...slider in the back aperture of the objective for the experiment press the 3D button Fig 108 Press again to deactivate If activated or deactivated the slider will not move immediately but only if a Sc...

Page 86: ...ters include all settings of the following tools Imaging Setup Acquisition Mode Channels Focus Devices and Strategy Z Stack Time Series Tile Scan Positions Regions Bleaching The blue block is the acti...

Page 87: ...the number of the Block in Start and End is run One loop repetition is set as default and reflects the minimum of running the experiment once Set the Loops value to 2 to perform the defined sequence t...

Page 88: ...the Macro ModifySeriesZEN lvb to combine auto saved images into consecutive time series images if needed See Chapter 2 MACROS AND VISUAL BASIC for further explanations Set desired stage position s for...

Page 89: ...objective that has a motorized correction ring an additional slider appears beneath the Objective selection Corr once this objective is selected The slider is used to set the correction ring to the b...

Page 90: ...the Andor iXon SMLM camera in the WF and Laser WF you have the options to select standard binning 512 x 512 standard 2 x binning 256 x 256 binned 2x2 or 4 x binning 128 x 128 4x4 All cameras are run i...

Page 91: ...Bit or 16 Bit Data Depth i e 256 4096 or 65536 gray values 5 2 8 Acquisition Parameter Incubation To have access to incubator functions you have to expand the Incubation tool by pressing the arrow in...

Page 92: ...Fit Gauss 2D box to use a full 2D Gauss fit routine or a Mask fit with a predefined fixed Gaussian mask if the box remains unchecked Fig 121 With the Peak mask size pixel slider you can adjust the siz...

Page 93: ...l tool tab how steep the activation power change will be The graphical display available only in the Show all mode will depict the shape of the ramp and displays the end laser power in If the initial...

Page 94: ...gs Defining Pinhole settings Defining Display options Expand the Channels tool window to have access to all options Fig 123 Fig 124 and Fig 125 Note that mixed mode tracks are not supported that is al...

Page 95: ...PTER 1 SYSTEM OPERATION ELYRA 7 Left Tool Area and Hardware Control Tools ZEISS 03 2019 V_02 000000 2262 999 91 Fig 125 Channels tool Appearance for the Laser WF imaging mode of ELYRA types S eLS syst...

Page 96: ...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 Track 1 and Track 2 a...

Page 97: ...e sequence is updated in the Imaging Setup tool accordingly You can add up to 4 tracks by pressing the button You can delete tracks by highlighting them and pressing the dust bin button The number of...

Page 98: ...ved 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...

Page 99: ...If a highlighted track is saved with a certain name or a predefined track with a name is loaded into a highlighted track the new name will appear in the Tracks designation box automatically Track desi...

Page 100: ...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 101: ...er becomes available that allows to set the laser power via the slider or the input box Note that for the WF imaging mode no laser line is available but the epi illumination is used Fig 138 Channel to...

Page 102: ...pressing either of the buttons EPI Epifluorescence HILO Highly Inclined Laminar Optical Sheet or TIRF Total Internal Reflection Fluorescence The active button is highlighted see Fig 142 You can also...

Page 103: ...step size for focus movement The value can be edited by typing or using the arrowheads The Home button sets the z position to the predefined zero value Pressing Load will lower the nose piece z positi...

Page 104: ...position of the microscope is changed via hand wheel or software the piezo objective focus is automatically levelled A click on the Leveling button moves the Piezo objective focus or Z Piezo stage ins...

Page 105: ...o control Use Z Piezo check box The Definite Focus tool allows control of the Definite Focus during continuous scan and for experiments were the Autofocus modus of the Focus Devices and Strategy is Of...

Page 106: ...te the Store Focus and the Recall Focus buttons greyed out buttons Even if the Periodic button stays active pressing the button will be ignored during the reflex finding run After the run all buttons...

Page 107: ...rupt the process you have to press the pressed Find Surface button during operation In this case Error will immediately be reported on the Status line and the run will end When a reflection is found t...

Page 108: ...us When pressing the Store Focus button Fig 155 the system will store the current sample Z position as the position the Definite Focus will stabilize on when pressing Periodic or Recall Focus There wi...

Page 109: ...the Periodic button is highlighted as pressed To stop periodic stabilization the highlighted Periodic button has to be pressed once again If the Definite Focus receives no or a too weak signal during...

Page 110: ...bilization the Status line will display Position not stabilized in red letters Fig 161 Pressing Recall Focus will interrupt a Periodic stabilization which will be resumed after the Recall Focus proces...

Page 111: ...input box displayed values and leaves the Piezo Position m input box values unaffected In contrast using the Z Piezo stage hand wheel will alter both display values The Z Piezo and the stand s Z driv...

Page 112: ...Focus no message is displayed in the information area which stays collapsed Fig 164 If the objective is unsuitable for Definite Focus or if there is no information available on its suitability the in...

Page 113: ...ing signal Stabilizing in blinking green whenever the Periodic button was pressed for continuous stabilization Stabilizing in green whenever the Periodic button was pressed for interval stabilization...

Page 114: ...Stabilization from the Focus Devices and Strategy tool runs synchronously to the scan In contrast stabilization from the Definite Focus tool will be asynchronous The state of the Use Z Piezo check box...

Page 115: ...the box to operate the Definite Focus in synchronous mode Fig 172 Define the cycles in number of frames in which the Definite Focus will be activated in the Autofocus every n Timepoint input box We s...

Page 116: ...essed the current center position of the image will be stored It can be recalled by selecting the position from the Marks position list drop down menu Fig 174 Pressing the Move to button will bring th...

Page 117: ...The Life Science cover glass 22x22 or the sample holder for TEM Grids should not be used together with Axio Imager and Axio Examiner equipped with a level adjustable sample holder During the calibrat...

Page 118: ...holder and fix it Life Science Cover Glass 22x22 Remove the clamping frame Fig 177 1 using tweezers Insert the cover glass Fig 177 2 in the sample holder Fig 177 3 Slide in the clamping frame into the...

Page 119: ..._02 000000 2262 999 115 Inserting a sample holder various mounting frames are possible Insert the sample holder into the mounting frame Fig 178 3 of the microscope stage in the following way for inver...

Page 120: ...for the calibration in the Marker Options field Move stage in load position before x y movement moves the stage to load position before moving to the next correlative calibration marker In case of us...

Page 121: ...r position was already set once and x y coordinates are given the stage can simply be moved to marker position one by clicking on the Position 1 button a Automatic marker recognition The software prop...

Page 122: ...arker on the sample holder is not recognized or user defined markers are used the marker positions can be set manually Activate the Acquisition Parameter Positions within the Acquisition Parameter lis...

Page 123: ...terest use the czi file format Enter the name of the image and click on Save Regions of interest ROI defined within the images will not be saved in the image format Mounting of the correlative sample...

Page 124: ...define Regions of Interest ROI which are used for display for WF SIM Apotome Lattice SIM and Laser WF imaging modes In widefield techniques WF SIM Apotome Lattice SIM Laser WF the whole sample is alw...

Page 125: ...on direction can be determined third click sets the second dimension and direction and ends the procedure Circle Drawing of a circle in the Image Display window click and keep the mouse button pressed...

Page 126: ...op down list of the Color mode The Color mode drop down menu allows defining how colors will be assigned to the outline of a region Fig 192 Individual colors allow the assignment of different colors t...

Page 127: ...ot available and will be ignored The numbers of the different regions will be shown in the image if the check box next to Show numbers is checked Regions can be saved by pressing the Save button using...

Page 128: ...n this way a 3 dimensional data set from the specimen is obtained The way in which the Z Stack is acquired can be selected by pressing either the First Last or Center mode buttons The modes differ in...

Page 129: ...otal Range may therefore vary to higher and lower numbers around the initial value of the Range Pressing the Opt Leap button only available in SIM Apotome and Lattice SIMimaging mode you can select be...

Page 130: ...he focus This tool might also be used for defining the first and last position for the Z Stack The F C and L buttons indicate the positions of the first slice center slice and last slice of the stack...

Page 131: ...sampling and accordingly adjusts the Slices to keep the Range within the limits defined by Set First and Set Last The optimal interval is displayed in the button in m Leap sets the spacing 3 fold broa...

Page 132: ...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 133: ...e of the Z Stack is limited depending on the type of Piezo Hence form the center position the Z Piezo stage can move half the total range each up and down Define the first and last slice using First L...

Page 134: ...requires an objective with a minimum magnification factor of 2 5x The number of Tiles can be defined in the Tiles Horizontal and Vertical Input boxes A maximum of 100 tiles can be set The size of the...

Page 135: ...re acquiring the tile scan When Bi directional is active available only in Show all mode the image acquisition is performed also on the horizontal backwards movement of the stage If the system is equi...

Page 136: ...rrent 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 using Add To this end click on the edge of the square t...

Page 137: ...ed area 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 ima...

Page 138: ...n be set The amount of data that can be handled by the system is limited If the maximum amount is exceeded a warning will appear In this case it is necessary to either change the destination for data...

Page 139: ...elay time will be effective for the time series acquisition Additionally it is possible to send a trigger signal out using the Trigger Out pull down menu at the start of each delay time Up to 4 Trigge...

Page 140: ...ion No to keep the configuration To save a configuration press the Save button symbolized by the disc icon This opens the Save current timer configuration as panel Fig 213 Type in an appropriate name...

Page 141: ...maximum of seven different markers can be defined using the button The button deletes the currently highlighted marker You can load a predefined timer configuration from the Marker field by pressing...

Page 142: ...configuration press the Save button symbolized by the disc icon This opens the Save current marker configuration as panel Fig 219 Type in an appropriate name in the Configuration field and press Ok to...

Page 143: ...set marker with time indication and comments blue automatically set marker with change of delay green automatically set marker at the beginning and at the end of a bleaching procedure 5 2 20 3 Start E...

Page 144: ...Scan checkbox is ticked Fig 224 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 145: ...me Series is finished If a trigger signal to end the scan arrives before the cycles have been processed the Time Series will only be interrupted Waiting for Trigger will be displayed in the status lin...

Page 146: ...and Strategy for details on offset values section Focus Devices and Strategies for Widefield Imaging Modes Highlighted positions can be removed by pressing the Remove button Alternatively the whole l...

Page 147: ...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 148: ...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 149: ...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 239 will display a help text how to calibrate the posit...

Page 150: ...ssing the Scan overview Image button opens the Scan overview Image interactive window Fig 241 Here you can define the number of Tiles by using the Horizontal and Vertical Input boxes the extension of...

Page 151: ...e current experiments in terms of Channels Bit Depth Dimension Required Disk Space Duration for 1 frame Any changes in the settings will be automatically updated in regard to the displayed parameters...

Page 152: ...ox is activated the Streaming tool is available Fig 244 Acquisition settings When Streaming is activated in the Streaming tool pressing Start Experiment will open a Windows Explorer window Fig 245 Her...

Page 153: ...l be available If you have an external processing PC you wish to stream onto make sure that the directory is mapped When you turn on the PC for system control after the external PC is completely boote...

Page 154: ...and analysis PC Most likely data transfer will be too slow and will jeopardize your experiment and will lead to an unstable system Do not use any USB devices external hard drives to stream onto data...

Page 155: ...osition etc preceding a number that indicates the frame number of the series If none is checked the whole image series will be saved in one file If the Sub directory checkbox is ticked in addition all...

Page 156: ...cess to all functions The Processing tool provides a number of methods for image processing and analysis using mathematical operators and algorithms which include Adjust Airyscan Processing Average Ch...

Page 157: ...MLM and Channel Alignment Processing tools are available which can recognize tables If you want to apply other Processing tools the SMLM image based on the table has to be converted to a true image Su...

Page 158: ...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 159: ...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 160: ...ction and use the slider or editing box to set a new value Fig 253 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...

Page 161: ...the dropdown list Fig 255 A 3D processing will take longer but will result in a higher resolution increase since also the axial Airy data will be used for the processing Use the same approach for R S...

Page 162: ...o hours depending on the number and type of files selected All files will be processed with the same processing strength and with the same mode 2D or 3D Do not mix the modes for processing as this wil...

Page 163: ...ng on an external workstation or storage computer This allows to process streamed data while acquisition is still running on the system computer Check External Processing to activate this functionalit...

Page 164: ...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 165: ...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 166: ...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 167: ...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 168: ...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 169: ...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 170: ...pixels set by the Strength slider It has an analogous effect as the kernel size Fig 267 If a dimension should be processed by this tool the respective X Y Z and Channels check boxes must be ticked The...

Page 171: ...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 172: ...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 173: ...acks only available in LSM imaging modes or multi channel images can be loaded In the Linear unmixing panel Fig 272 the number of spectrally distinguishable fluorescent components within the imaged sa...

Page 174: ...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 175: ...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 176: ...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 177: ...entrations in physiological experiments To open the Ion Concentration tools click Ion concentration Fig 276 The two buttons select the active image in the Image Display as Input Image and Input Image...

Page 178: ...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 179: ...1 Single Wavelength Dyes Offline Calibration Subtract background autofluorescence image from raw images to obtain better raw data to start with Fig 279 Perform equation or titration calibration compar...

Page 180: ...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 181: ...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 182: ...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 183: ...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 184: ...he Modify mode drop down menu Fig 285 and Fig 286 It allows modifying the acquisition date rotating or mirroring the image s converting or reversing dimensions renaming channels or making time series...

Page 185: ...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 186: ...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 187: ...display This step visibly intensifies weak signals Fig 289 Unlike the Sampling method the Illumination method of HDR still provides a linear grey stepping which allows quantitative measurements For t...

Page 188: ...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 189: ...nction tiled 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 292 The Correl...

Page 190: ...alization Miscroscopy tools Fig 293 is applied to data acquired in the SMLM imaging mode It subdivides in 4 subcategories Convert to Image Converts the table vector map of a SMLM image to a bona fide...

Page 191: ...ect the Convert to Image processing tool Fig 294 Highlight the SMLM image in the image container you want to convert and press Select Fig 295 Press Apply to convert the HR channel to a true image The...

Page 192: ...molecules tab to select the Import SMLM molecules processing tool Fig 296 Press Select Fig 297 to open Windows Explorer and select the molecule table you want to import Press Open to load the table a...

Page 193: ...ote that the localization 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 dep...

Page 194: ...d Position threshold 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 SMLM image This tool will emph...

Page 195: ...e actively selected image The Channel drop down menu allows you in a multi channel experiment to apply different settings to different channels which are all listed Fig 308 If you select All all chann...

Page 196: ...due to the Maximum cluster size setting The Dimension drop down menu will display the dimensionality 2D or 3D of the selected SMLM image which is automatically recognized if a czi format is loaded Fi...

Page 197: ...the circles will correspond to the set value of the Peak mask size Fig 315 The bigger the radius the more prone are closely juxtaposed peaks to overlap The smaller the circles the more likely it is t...

Page 198: ...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 199: ...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 200: ...alculated 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 pre...

Page 201: ...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 particl...

Page 202: ...ot influence the Preview image Note that using Grouping in the processing tool is irreversible Therefore in the created SMLM Grouping tab all functions will be greyed out and not accessible If all par...

Page 203: ...e Method Parameters tool window to have access to the Particle Tracking tools Fig 328 You can set different parameters to define the molecules and the calculation of the trajectories Peak intensity to...

Page 204: ...200 000000 2262 999 03 2019 V_02 Once the settings are complete press Apply to obtain the trajectories of the single molecules Fig 329 Please note that Particle Tracking only works for 2D data sets Fi...

Page 205: ...he Experimental PSF by choosing Automatic from Mode drop down menu Fig 332 If you want to process manually and set quality parameters select Adjust Parameters Fig 333 If you want to edit selected bead...

Page 206: ...the original image 3 pixel size of the resulting PSF is 3 times smaller as in the original image 5 pixel size of the resulting PSF is 5 times smaller as in the original image or 7 pixel size of the r...

Page 207: ...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 208: ...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 209: ...l set for details The Processing SIM tools works exclusively for data acquired in the SIM Apotome and Stripe Lattice SIM imaging modes with the exception of Stripe SIM tool that only works for Stripe...

Page 210: ...setting parameters Fig 341 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 211: ...ntly saved parameter values will be loaded With the Channel selection drop down menu you can choose All channels or the channel you want to process if the image has more than 1 channel Fig 345 The Cha...

Page 212: ...ads are sorted according to these characteristics resulting in 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...

Page 213: ...ity due to the Talbot effect whereas the 2nd and higher orders should be more or less constant over the whole z range As an output there will also be two files in the C Temp folder called ModulationCo...

Page 214: ...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 defined in the image t...

Page 215: ...k that should be processed Fig 352 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 353 With the Mode drop down menu you h...

Page 216: ...nd parameters become accessible to be changed and the Manual selection field becomes accessible Fig 357 The Automatic mode is recommended for images with high modulation contrast In case the modulatio...

Page 217: ...mal negative intensity becomes zero intensity baseline shifted In Baseline Cut negative values will be clipped by setting them to zero No offset is applied The Sectioning or Strength filter removes th...

Page 218: ...you have to check the Raw scale box In this case the orifginal dynamic range is kept You can select between theoretically computed or experimentally determined PSF by choosing Theoretical or Experime...

Page 219: ...n for Input image 2 Then expand the Methods Parameters tool window to have access to setting parameters Fig 367 You can toggle between Input image 1 and 2 in the Preview tab window which also displays...

Page 220: ...ys all channels of the SIM Input Image Fig 369 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 221: ...The Processing SIM tool works exclusively for data acquired in the SIM Apotome as well as Stripe Lattice SIM imaging modes The SIM function allows automatic or manual processing of a set of structure...

Page 222: ...s contribution will be used in addition to the information of the frame itself This will result in better x y and z resolution and better contrast by removing the out of focus light to a greater exten...

Page 223: ...tem will display the SIM image for this ROI only Moving the ROI will update the SIM image on the fly This might help to fast evaluate the fitted parameters and make adjustments for the processing of t...

Page 224: ...for Batch processing You can instead of processing only one selected image also load more images to be processed sequentially To this end press the Run Batch button Fig 381 This will open Windows Exp...

Page 225: ...t contrast in z Best Fit Parameters are fitted by using the whole image In the Advanced Filters field a Detrend check box is available that becomes active for time series data only If checked the algo...

Page 226: ...e box In this case the original dynamic range is kept You can select between theoretically computed or experimentally determined PSF by ticking or unticking the Experimental PSF check box Fig 389 If n...

Page 227: ...o the alignment tools Fig 390 Activate the first image you want to process in the image container and press the Select button for Input image to load the image If you want to load a second input highl...

Page 228: ...different for SMLM images The high resolution HR and sum widefield SWF channels are also listed alternatively with the HR channels preceding their corresponding SWF channels however the format of the...

Page 229: ...ou can set a ROI and then the structure within the ROI is used For HR SMLM images at least three fiducials are expected Without fiducials no alignment will be performed in this case Parabolic is used...

Page 230: ...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 397 Press Apply to extract that image from the time series...

Page 231: ...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 400 The number of channels depend on the number of channels of...

Page 232: ...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 233: ...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 234: ...on with Time Series When a dataset has been acquired using Dual Side illumination the dataset will have the illumination dimension saved in its metadata and the Dimension slider for Illumination is vi...

Page 235: ...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 236: ...0 2262 999 03 2019 V_02 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 237: ...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 238: ...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 239: ...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 240: ...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 241: ...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 242: ...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 243: ...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 244: ...ve the same size If the scaling in Deconvolution PSF settings Fig 409 is changed the latter will override the scaling settings here Individual Registration For Every Time point If the multiview experi...

Page 245: ...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 246: ...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 247: ...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 248: ...the same size 5 3 21 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 249: ...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 250: ...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 251: ...Left and Right as red and blue lines respectively Fig 421 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 252: ...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 253: ...ested 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...

Page 254: ...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 255: ...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 256: ...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 257: ...sis and Remove structures Fig 429 5 3 23 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 258: ...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 259: ...window The correlation to be computed can be selected from the Correlation drop down menu Fig 432 For one channel recordings only Spatial auto correlation S A is available for a two channel recording...

Page 260: ...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 261: ...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 speed needs to be ada...

Page 262: ...nd the much slower movements 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...

Page 263: ...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 264: ...5 3 24 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 265: ...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 266: ...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 267: ...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 268: ...is not a time series the Select button stays inactive Choose how you want to detrend the data If the Refer to Previous checkbox is ticked the software detrends the data of a frame in reference to the...

Page 269: ...p allow the calibration and adjustment of hardware settings The following options are available Dual Camera Adapter Alignment Define Objectives Adjust SMLM Slider only available for systems with 3D SM...

Page 270: ...ckbox is ticked Fig 445 Inthis case no Continuous Scan or laser light iis needed as an internal LED lamp will be used for illumination the pattern Press Adustment XY Adjustment XYRot or Adjustment Z t...

Page 271: ...ce the objective is activated its focus speed and parfocality can be set 5 4 2 1 Detection Objective Press the Detection Objective tab to have access to the Objective definition window Fig 447 Press a...

Page 272: ...jectives 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 448 or Potential Objectives s...

Page 273: ...d 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 274: ...he objective from No to keep it in the database or Cancel to abort the process 5 4 2 1 4 Show Edit Objective This function allows viewing the parameters of an objective from all objective directories...

Page 275: ...sing the Write button the new settings will apply for computer and manual control 5 4 2 3 Parfocal Correction Press the Parfocal Correction tab to have access to the Parfocal Correction definition win...

Page 276: ...e by pressing Next The Next button only becomes available when the Start button is pressed If Next is pressed the next objective in the nosepiece is selected and the parfocal adjustment performed Pres...

Page 277: ...e objective in use by pressing the Store button or for all objectives by pressing the Store for all objectives button The saved positions will be used whenever the respective objective is in use There...

Page 278: ...and will be in the middle Fig 463 EPI illumination using 100 nm beads sample and using Bertrand lens to look into the back aperture og the objective Use Tube lens 1x Move the slider until it divides...

Page 279: ...unctions by sliding the tabs to the left and right directions The downwards pointing triangle will list all the Setting functions in a drop down menu Fig 466 Selecting a function will automatically ac...

Page 280: ...or the AxioCam Mrc 3 0 camera from the vertical shift speed drop down menu Fig 469 This will affect the speed the camera chip is read out vertically Select the horizontal shift speed not available for...

Page 281: ...parameters 5 4 4 2 System Options Elyra Press the Elyra tab to open the Elyra panel Fig 471 Tick the Online pre processing SR SIM check box to activate pre processing of SIM data already during image...

Page 282: ...oaded The following parameters are available Objective Frame size Speed Data depth Scan direction not applicable Average Pinhole diameter not applicable Detector gain and offset Zoom Rotation and offs...

Page 283: ...ig 475 If the Apply brightness and contrast changes to diagrams and tables is ticked any changes in brightness and contrast of the image will also affect the image data shown in diagrams or tables If...

Page 284: ...unless this function is active Note When a file set is opened via the masterfile and modified i e cut region subset etc and then saved again the new data set will be saved only in one file and not in...

Page 285: ...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 286: ...ctivated 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 so th...

Page 287: ...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 288: ...section Processing Copy Image Handling and Container Layout Configuration The control area Fig 483 on the top right corner of the Image Display is for scrolling click on the left or right triangle but...

Page 289: ...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 290: ...dimensions of the image data set The sections slices in the dimensions can be scrolled with sliders Z Position and Time in Fig 487 and also directly addressed with setting numbers in the spin boxes ne...

Page 291: ...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 292: ...Acquisition Mode Click on will display the Crop Rectangle in the Image Display Fig 491 Any changes done with the Crop Rectangle are setting the scan parameters immediately On the next execution of a s...

Page 293: ...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 294: ...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 295: ...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 296: ...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 297: ...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 298: ...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 299: ...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 Zoom All button is added syn...

Page 300: ...d SWF channel images Fig 499 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 c...

Page 301: ...nnels belonging to the same color will be merged in one image Fig 502 If group modes is selected all channels belonging to one mode will be merged in one image Fig 503 Fig 502 Image Display Split view...

Page 302: ...the Dimensions Display Player and Graphics View Options control blocks apply The additional view specific Gallery View Option control block includes a tick box to switch on and off the display of the...

Page 303: ...CHAPTER 1 SYSTEM OPERATION ELYRA 7 Center Screen Area Image Containers Display and Image Analysis ZEISS 03 2019 V_02 000000 2262 999 299 Fig 504 Image Display window Gallery display...

Page 304: ...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 305: ...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 306: ...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...

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Page 311: ...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 312: ...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 313: ...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 314: ...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 315: ...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 316: ...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 317: ...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 318: ...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 319: ...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 320: ...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 321: ...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 322: ...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 g...

Page 323: ...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 324: ...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 325: ...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 326: ...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 327: ...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 328: ...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 329: ...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 330: ...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 331: ...M OPERATION ELYRA 7 Center Screen Area Image Containers Display and Image Analysis ZEISS 03 2019 V_02 000000 2262 999 327 Fig 543 Image Display Histogram view The Histo button can also be used online...

Page 332: ...te values Show Image tick box Shows the image in the Image Display window with the histogram graph The Cut Mask tool creates a new image document which sets every pixel outside ROIs to Zero Within the...

Page 333: ...n the context of digital imaging the term colocalization refers to colors emitted by fluorescent molecules detected by the same pixel in the image It is important to be aware of the fact that colocali...

Page 334: ...OPERATION ZEISS Center Screen Area Image Containers Display and Image Analysis ELYRA 7 330 000000 2262 999 03 2019 V_02 Fig 546 Image Display Colocalization view Fig 547 Scatter diagram and threshold...

Page 335: ...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 336: ...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 337: ...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 54...

Page 338: ...SS Center Screen Area Image Containers Display and Image Analysis ELYRA 7 334 000000 2262 999 03 2019 V_02 Fig 549 Image Display Profile View Line Profile with markers Fig 550 Image Display Profile Vi...

Page 339: ...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 340: ...ined regions and points are no longer valid Fig 551 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 341: ...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 342: ...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 343: ...n of ROI View Option control block is shown in Fig 552 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 344: ...en Area Image Containers Display and Image Analysis ELYRA 7 340 000000 2262 999 03 2019 V_02 Fig 553 Image Display window Mean ROI display for time series in single plane Fig 554 Image Display window...

Page 345: ...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 346: ...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 347: ...ay Player and Graphics View Options control blocks apply The additional view specific FRAP View Option control block is shown in Fig 555 Any changes done with this View Option control block are effect...

Page 348: ...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 349: ...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 350: ...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 351: ...age Containers Display and Image Analysis ZEISS 03 2019 V_02 000000 2262 999 347 Fig 558 Image window displaying the analysis of FRAP data using a mono exponential fit Fig 559 Image window displaying...

Page 352: ...he 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 protein...

Page 353: ...t emission filter see Fig 562 The settings of the Graphics View Options control block apply as in 2D view In the Display View Options control block the channel specific settings of brightness contrast...

Page 354: ...and the functionality of the four general View Option control blocks Dimensions Display and Graphics is the same as in the Lambda Coded View see the previous section on this view type The two additio...

Page 355: ...tic Component Extraction ACE can be added Select ROI for background subtraction Automatically balances the intensity of unmixed channels to equal levels A statistical analysis of the signal related Po...

Page 356: ...ction Automatically searches the image for regions with pure signals Fluorescence originating from one single dye Performs the unmixing with the selected spectra Note Those channels of the Lambda stac...

Page 357: ...e unmix the data set by clicking the button Fig 567 A new and spectrally unmixed multichannel image document is created as many channels as previously defined components Benefit and limitations of the...

Page 358: ...d by right mouse clicking on the table display Fig 568 FRET Display of Image after Sensitized Emission analysis After a Sensitized Emission analysis the table shows the following parameters Fig 568 Re...

Page 359: ...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 360: ...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 361: ...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 362: ...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 363: ...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 364: ...f the parameters used for the image analysis The General Fig 573 tab applies to both methods Acceptor Photobleaching and Sensitized Emission If checked the listed options will be taken into account fo...

Page 365: ...iew control tabs respectively or only the merged SMLM and sum widefield images for Offline Processed images Fig 579 For 3D SMLM images only the processed HR image is available A high resolution SR and...

Page 366: ...ion the dimension will be displayed as Z Position and with the Z Position slider you can scroll through the data set Fig 583 Please note that the spinbox display shows the number of the current slice...

Page 367: ...is no distinction between Source and SMLM image In the Modify field you can press Source Image or SMLM image If Source Image is pressed the TV channels time series become available in the Channels fi...

Page 368: ...tool Copy Two or more channels of the SMLM image mixed mode with HR and SWF channels can either be obtained by processing a 2 color channel Source image in the Processing tool SMLM or by using the Pro...

Page 369: ...ntainers Display and Image Analysis ZEISS 03 2019 V_02 000000 2262 999 365 The Group drop down menu allows you to assign the way the mixed modes channels can be grouped Fig 591 Fig 590 Dimension view...

Page 370: ...idually HR1 HR2 etc single SWF1 SWF2 etc single Fig 592 group channels channels are grouped according to their modes HR1 HR2 etc together SWF1 SWF2 etc together Fig 593 group modes channels are groupe...

Page 371: ...ge or SMLM image If Source Image is pressed the TV channels time series become available in the Channels field Fig 596 Fig 597 If only one TV channel is available no channel buttons are displayed for...

Page 372: ...ics View Control Tab The selection and function of regions of interest ROI have been described in section 2D View Fig 600 All ROIs selected in Graphics will be used for reconstructing a SMLM image if...

Page 373: ...le in the Show all mode Fig 601 You can select between Fiducials drift correction will be performed on fiucial markers in the image Fig 602 Model based drift correction will be performed on sample str...

Page 374: ...ive Fiducials selected as Correction type Fig 605 SMLM Drift view control tab of the SMLM view tab with Show all mode not active Model based selected as Correction type Please note that Fiducials and...

Page 375: ...arded as a fiducial Capture radius pixel Sets the maximum radius around an object where another object is tolerated in order that the object is regarded as a fiducial When Apply is pressed the softwar...

Page 376: ...he button again the table will disappear The table has different columns Use If the box is checked the corresponding fiducial is selected and will be used for drift calculation If the box is not check...

Page 377: ...s by crosses and a circle around the localizations The fiducial highlighted in the Fiducials Table has a circle with a thicker line Not all positions of a fiducial are displayed in the overlay just ev...

Page 378: ...ducial and click into the image with the left mouse button The fiducial at the selected position will be added to the table To inactivate the button press it again You do not need to have the fiducial...

Page 379: ...ice versa if the drift diagram was active and the Drift Table button is pressed the drift diagram will be replaced by the drift table and the Drift Diagram button will not be active any longer The dri...

Page 380: ...t the zoom if the diagram was zoomed Copy diagram Copies the diagram into the clipboard Save diagram Saves diagram as a tagged image file tif CompuServe GIF gif or JPEG file interchange format jpg fil...

Page 381: ...splay and Image Analysis ZEISS 03 2019 V_02 000000 2262 999 377 With the left mouse button pressed you can by drawing a rectangle zoom into the defined region of the diagram Fig 622 Fig 621 Drift Diag...

Page 382: ...ing the active Correction button it becomes inactive and the image will be displayed without the drift correction applied The drift correction is hereby not undone in the molecule table only in the di...

Page 383: ...quals number of z drift controls plus 1 If no z drift correction was performed the number 1 is displayed which than will be used as the number of segments Fig 625 Segment drop down menu with Automatic...

Page 384: ...ted and the drift diagram becomes available Fig 629 By pressing the button again the drift diagram disappears The drift diagram displays the drift in the x and y direction as separate graphs versus th...

Page 385: ...iagram Copies the diagram into the clipboard Save diagram Saves diagram as a tagged image file tif CompuServe GIF gif or JPEG file interchange format jpg file Copy diagram data Copies the diagram data...

Page 386: ...ift correction in the y direction in nm Drift Z nm Drift correction in z direction in nm for 3D SMLM data set only By pressing the right mouse button into the Fiducials Table or the Drift Table a cont...

Page 387: ...ed to belong to the same molecule If the Group button is pressed peaks are grouped to one molecule according to the set parameters Since peaks are summed the grouped image will have fewer objects Fig...

Page 388: ...Press Yes to overwrite In this case grouping and drift correction cannot be reversed or altered if the saved image is loaded later The controls in the SMLM Drift and SMLM Grouping view control tabs w...

Page 389: ...grouped data Fig 640 or object for grouped data Fig 641 will be displayed By pressing the highlighted Table button the molecule table will disappear and the button will not be highlighted any more A v...

Page 390: ...n nm in x y Precision z nm Calculated localization precision of a peak object in nm in z Number Photons Total number of photons of a peak object Background variance Variance of the number of backgroun...

Page 391: ...87 If Histogram is selected the histogram specific functions will be displayed Fig 644 With the Histogram Source drop down menu you can select the parameter that should be plotted in the histogram Fig...

Page 392: ...will be set for the data There a histogram will be rescaled Fig 647 in a way that only those x values are displayed that accommodate the input of the data starting from 0 Hence outliers with high valu...

Page 393: ...IF gif or JPEG file interchange format jpg file Copy diagram data Copies the diagram data table into the clipboard Save diagram data Saves the diagram data table as a text txt file If Scatter Plot is...

Page 394: ...left mouse button Fig 654 By clicking with the right mouse button into the diagram the Histogram context menu becomes available Fig 655 You can choose between the following options Reset zoom Will res...

Page 395: ...these values are frame wise updated The following parameter values are displayed Position X m Position in m of the selected spot in the X direction with the origin at the upper left corner of the imag...

Page 396: ...ice versa if the peak tracking was active and the Table button is pressed the peak tracker will be replaced by the molecule table and the Peak Tracker button will not be active any longer 6 18 7 SMLM...

Page 397: ...tion PSF calculated from the Gauss fit only available for 2D SMLM data sets min 1 max 500 Background Variance of the number of background photons min 1 max 5000 only available in Show all mode Chi squ...

Page 398: ...SMLM Filter settings will be applied to images acquired during Online Processing All SMLM Filter settings will be independently applied for each slice of the Z Slice Please be aware that the last filt...

Page 399: ...b 6 18 8 1 Rendering Data Sets In the SMLM Rendering view control tab the display of the data can be set Fig 662 Different settings are available for 2D SMLM Fig 662 or 3D SMLM Fig 663 data sets Fig 6...

Page 400: ...o Nyquist the pixel or voxel size must be at least half of the displayed localization precision and resolution in order to obtain the displayed localization precision Localization precision LP is not...

Page 401: ...areas with higher molecule densities are also brighter Centroid Localized peaks objects will be displayed by a dot at the localization point Gauss Centroid Overlay of Gauss rendered peaks objects and...

Page 402: ...Mode Color drop down menu is available Fig 670 Fig 671 from which the color of the overlay centroid or cross can be selected Fig 672 For all Display Modes displaying a Gauss rendered peak object Gauss...

Page 403: ...x is checked the Expansion factor is no longer available For all Display Modes displaying a rendered peak object and for density maps Gauss Gauss Centroid Gauss Cross Molecule Density the Render autod...

Page 404: ...es the dimmer parts get brighter if autodynamic range is applied In case of the SWF image the brightest spots will get into saturation For all Display Modes displaying a density map Molecule Density t...

Page 405: ...D SMLM data have been processed with an Experimental PSF without attached Localization Precision Look Up Table LUT events will be displayed as dots which can be vizualized larger using the Render Loca...

Page 406: ...he Split view tab both the HR and the SMW image if both selected can be viewd sied by side as two channels Fig 682 For 3D SMLM data you have several options to explore the axial dimension 2D view with...

Page 407: ...u can scroll the z slices with the Dimension Z Position slider Fig 683 The z position can also be dsiplayed as a color map were color hue codes for z position using the Processing function Color coded...

Page 408: ...olor code bar to alter its size by drawing on the corners format by drawing on the corners and changing the extension and position by drag and drop Font appearance and size can be changed under More o...

Page 409: ...l might help 3D view allows you to take distance measurements As 2D and 3D SMLM images are vector maps which are not recognized in many Processing functions and bigger in their size than normal images...

Page 410: ...time series Besides the general control view tabs Dimensions Display and Graphics available which functions have been described in section 2D View there is a special Particle Tracking view tab availa...

Page 411: ...ists all particles identified with the set minimal Trajectory length of which the track is displayed in the image It has for columns Index Identifies the Particle with a number First frame The frame t...

Page 412: ...d 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 the following way r r P 2 3 and P P r 3 2 In a c...

Page 413: ...d 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 plane time series Tile scan information The zoom facto...

Page 414: ...CHAPTER 1 SYSTEM OPERATION ZEISS Center Screen Area Image Containers Display and Image Analysis ELYRA 7 410 000000 2262 999 03 2019 V_02 Fig 691 Information View of a processed superresolution image...

Page 415: ...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 416: ...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 417: ...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 418: ...03 2019 V_02 Within the dialog Fig 696 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 419: ...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 420: ...CHAPTER 1 SYSTEM OPERATION ZEISS Right Tool Area Data Management and Storage ELYRA 7 416 000000 2262 999 03 2019 V_02 Fig 699 Images with information...

Page 421: ...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 422: ...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 702 are available The...

Page 423: ...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 424: ...o ZEN blue edition Not all image versions can be opened in ZEN blue edition Data acquired with ELYRA might not be displayed in the right way by ZEN blue edition Regardless of which display type is cho...

Page 425: ...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 708 The stars...

Page 426: ...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 427: ...be exported to Fig 711 Select the data type which the image is to be exported under Data Fig 712 Chose a compression level For some file formats lossless compression or various other compression leve...

Page 428: ...Auto Save 150 Automatic component extraction 353 Average 164 Averaging selection 87 B Band filter 167 Beam path configuration 71 Big view button 48 Black 289 Blending 249 Brightness 260 C Calibration...

Page 429: ...ture 254 ICS correlation 255 Image Export of an 423 Saving an 422 Image calculator 162 Operators 163 Image control area 45 Image information 48 Image tabs 45 Image view controls 45 Image view tabs 45...

Page 430: ...e document button 47 O Objective 267 Objective selection 75 85 Objectives 267 Offline calibration 175 Online multiview processing 250 Online ratio 176 Options 275 Options tool group System Options 275...

Page 431: ...89 Remove PSMLM outliers 190 SMLM view 361 Smooth filter 166 Snap 63 Start 139 Start experiment 65 Starting ZEN 20 Startup 19 Statistical confidence 171 Status bar 39 Stitch 185 Streaming 148 149 159...

Page 432: ...CHAPTER 1 SYSTEM OPERATION ZEISS Index ELYRA 428 000000 2262 999 03 2019 V_02 Workspace zoom 31 Z ZEN File Browser 33 412 Form view 417 Gallery view 415 Table view 418 Z Piezo stage 129 Z Stack 124...

Page 433: ...7 2 3 Overview of Available Macros 8 2 4 Sample Macros 9 2 5 ExcitationFingerprinting 9 2 6 ConcatenateImages 12 3 Object browser 13 4 COM interface 14 5 Glossary 14 6 Programming with the VBA GUI in...

Page 434: ...s bipartite Macro editing and recording of macros assign macros to list Visual Macro Editor edit automatic workflows with pre defined modules Selecting Macro opens a dialogue for editing and recording...

Page 435: ...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 436: ...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 437: ...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 438: ...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 439: ...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 440: ...Z Stacks and Time Stacks like Rotation of the stacks being mirrored Conversion of time stacks into Z Stacks and vice versa A more detailed description is available within the macro folder as separate...

Page 441: ...er for Excitation fingerprinting it is recommended to calibrate for the average power at the sample plane using a power meter The resolution of the power meter should be below 1 mW For the calibration...

Page 442: ...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 443: ...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 444: ...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 445: ...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 string ex...

Page 446: ...mple to connect a MATLAB session to ZEN start ZEN first start MATLAB and then type ZENVBA actxGetRunningServer Lsm5Vba Application ZEN actxGetRunningServer Zeiss Micro AIM ApplicationInterface Applica...

Page 447: ...ind the 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 th...

Page 448: ...to read 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 do...

Page 449: ...t can be obtained directly from here 6 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...

Page 450: ...EN Macros ExampleMacros backup_recording lvb 7 3 Event handling Events can be used to react to actions by the user or by the hardware Typically events in ZEN are either Mouse events Scan state events...

Page 451: ...ZEISS 03 2019 V_02 000000 2262 999 1 CHAPTER 3 ADDITIONAL SOFTWARE TOOLS CONTENTS Page 1 System Maintenance Tool 2 1 1 Calibration Objective 2 1 2 Starting the Maintenance Tool 3 1 3 General Operation...

Page 452: ...selected tool for Service 1 1 Calibration Objective Observe the following notes The calibration objective is not suitable for multiphoton excitation there is a risk of bubble formation in the substrat...

Page 453: ...xisting lenses the calibration objective needs 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...

Page 454: ...omer type in any name not available The Maintenance Tool can only be used if the calibration objective is entered as APO Calibration LSM If this is not the case a message will remind the user to selec...

Page 455: ...ask requires any manual adjustments the Maintenance Tool guides users to all adjustment steps After the completion of a task the result is displayed with a traffic light type green yellow red indicato...

Page 456: ...CHAPTER 3 ADDITIONAL SOFTWARE TOOLS ZEISS System Maintenance Tool ELYRA 7 6 000000 2262 999 03 2019 V_02 Fig 8 The Maintenance Tool guides users through required adjustment steps...

Page 457: ...database 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...

Page 458: ...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 459: ...rring to the detection channel it is effective for Fig 10 Clicking this button opens the Change Filter window with a list of favourite potential and user defined filters Fig 12 To define a new filter...

Page 460: ...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 461: ...luorescence 16 5 3 Stage Leveling Using X Cite in Reflection 18 5 4 Stage leveling using X cite in reflection and two glas plates 20 6 Liquid Cooling System of ELYRA 21 7 Autocorr Objectives Installat...

Page 462: ...e VBA Software ZEN Module Shuttle Find Software ZEN Module Experiment Designer Software ZEN Module 3D VisArt Software ZEN Module SIM Software ZEN Module Tiles Positions Software ZEN Module FRET If you...

Page 463: ...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 your configuration does not include...

Page 464: ...of photomultiplier Transmitted light PMT Non Descanned Detectors NDD If your configuration does not include the Piezo objective focusing device or the Z Piezo stage the following functions are not ava...

Page 465: ...em in an Optimized Way ZEISS is offering training courses on how to operate the system in an optimized way Courses are held in our application center in Jena Germany Courses are held in English or Ger...

Page 466: ...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 precis...

Page 467: ...M27 Installation Screw in your microscope objective into Piezo Objective Focusing Device see Fig 1 1 Screw the thread ring into your microscope see Fig 1 2 Easy clamp the Piezo Objective Focusing Dev...

Page 468: ...ignals 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 469: ...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 470: ...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 471: ...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 472: ...the user interface on the realtime electronics Fig 2 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 473: ...ts of the microscope e g the condenser unit When moving a motorized stage avoid any collisions Do not install inserts while the stage is moving Be careful when switching the condenser front lens 5 1 L...

Page 474: ...hen changing samples it is sufficient to take out only the sample holder which is most conveniently done by pulling on the shafts of the upper and lower spring clamps Fig 3 uss lss The strength of the...

Page 475: ...use the springs can easily be stored by bringing them in a parallel position to the sample holder s longer edge Secure in this position by pressing the spring down The three point mounting allows for...

Page 476: ...ustment the incubator has to be closed to monitor the outcome and the adjustment has to be done iteratively The level adjustable stage insert can become helpful when working with water objectives as t...

Page 477: ...curate if the sample was positioned in the center d Use the back left screw Fig 6 sx of the sample holder to readjust the focus Fig 7 E Turn clockwise if the focus position was positive counter clockw...

Page 478: ...pty position and use preferentially Andor SIM camera albeit the Andor PALM camera would also work Take Optovar 1 0 and set EM gain to zero 2 Draw fiber plug of X Cite completely out from the X Cite ca...

Page 479: ...llows X Xmax X Y Ymax Y with Xmax and Ymax representing the number of pixels of the camera in X and Y direction E g if the coordinates X and Y would have been 502 and 512 respectively the new coordina...

Page 480: ...edge camera albeit the Andor PALM camera would also work Take Optovar 1 0 and set EM gain to zero 3 Draw fiber plug of X Cite completely out from the X Cite casing Start acquisition The image will be...

Page 481: ...constant System is working Red LED flashing once Cooling liquid is too warm Check the temperature in the room It should be at 22 C plus minus 3 C If it is more than 30 C the cooling system cannot wor...

Page 482: ...fits without colliding Don t forget to retighten the screws Connect the objective to the can distributor box using any free connector Restart the microscope system Open the ZEN Configuration Tool and...

Page 483: ...offer Cuda compatible graphics boards The system installation and OS is located on a redundant RAID on most of our computers In case the speed of the RAID is not optimal for OS file swap transfer a f...

Page 484: ...D or 10GbE card 8 4 Networking of ZEISS Microscopy Computers ZEISS microscope computers can be networked The main purpose of such networking is storage of image data faster processing workspace econom...

Page 485: ...allow connection to a campus network Optional 4TB HDDs allow to increase the shared storage space of the computers co and comparable 8 6 Solution 2a Storage Shielded In this storage PC configuration I...

Page 486: ...itself is also open to the campus network Optional HDDs allow to increase the storage space of the computers in addition to the storage PC co and comparable 8 8 Solution 3 Dual Storage In this big dua...

Page 487: ...eous peer to peer configuration a number of Imaging microscopes with and without workstation PCs are networked via WiFi USB and LAN connections Images can be viewed via iPads or analysed on one of the...

Page 488: ......

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