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Andor iQ USER GUIDE

1. more details Keyword Icon Hint Click below Auto Focus RA Execute Autofocus action Autofocus Auto Focus E Specify frequency of Autofocus Autofocus Activate Activate action AutoSave Not strictly in Protocol Tree but Auto Save related Camera a Set camera binning to specified Camera Binning Binning value Camera gt E Select camera for current Camera Selection selection Protocol Delay da Wait here for defined period d Delay End x i End of iterator loop type x see End Repeat x End x can be dragged downwards to bracket other Actions Frappa Wr Execute a Photo stimulation Frappa action Image BE Root name of the images Image created by the Protocol Load Protocol pi Load and execute Protocol at Load Protocol End of current Protocol Montage View ke Show and execute Montage Montage View View Move Channel NO Set devices to state recorded in Move Channel Channel Move Channel Ao Set devices to state recorded in Move Channel Snap AND Snap Channel and capture a frame Move XY RX Set XY stage to specified Move XY position with Z Move Ref Z amp Set Reference Z stage to Move Ref Z specified location 193 Andor Technology Andor iQ USER GUIDE Move Scan Z OnLine Chart Pause Protocol Type Ratio Rep
2. es Please enter a name for the new Scan explained later L r eaea Note BEFORE you define a multi well scan Options MultiWell Petri Scan pattern or montage scan you MUST f Systematicaly sampled fields C User defined fields successfully run the Stage Alignment Multifield Scan Wizard Montage Press Next to proceed to the next Zseries step File ImageList Settings QC Wizards Plug Ins Help Setup Wizard Mult Vell Petri setup Well settings es Select Create new well pattern then 2 gt press Next to continue lt Back Neo Cancel Note fyou have previously created a Select Pattern Select one of the following options well pattern you can Use existing well C Use existing well pattern pattern or Edit existing well pattern to modify its parameters Edit existing well pattern If you want to apply the same Well pattern but for a different experiment then use Edit 168 Andor Technology Andor iQ USER GUIDE ee ee eee ae From the dropdown list box on the etup Wizard Kaulti vy ell Petri dialog red highlight select the type of setup Well settings scan required lt Back Next gt gt Cancel setup New Scan Enter a unique name for the scan Enter a name forthe new pattern pattern into the text box Scan patterns Costar SSF 3xz 35 29 This name must be unique as it will be used as the filename when the well patter
3. Process Status Ibl_Message 331 Andor Technology Andor iQ USER GUIDE 5 12 2 Montage View Options The Options dialog allows you to choose how image edges are merged Brightest is best for fluorescence images lf Pixel Matching is checked then iQ attempts to match image edges by correlation matching regions to find a best fit Based on the best correlation iQ will stitch images with the correlation peak offset in X and Y You can decide how large a range iQ should search over to match pixels and in the drop down box where you can select 1 5 and 10 um by default but you can add further values depending on your specific configuration and results You can also specify which channel is used for matching Montage View C DOCUMENTS AND SET TINGS W BROWNEWESKTOP IQ IMAGES MFMONZT2CH Source DESKTOPUQ IMAGES MFMONZT2CH 2x2 16102009 T1F Sampling Factor f WV Update Each Frame Show Cancel ole Options Process Status Rendering Field 98 of 900 To produce the montage result in the shorted time leave Pixel Matching unchecked In this case edge matching will be limited by stage precision So the choice depends on your needs lf you check Update Each Frame the image will be redrawn for each frame tiling Andor Technology Andor iQ USER GUIDE operation as shown above This will slow down reconstruction but it provides useful feedback The Montage dialog above shows the result being update
4. Settings ere When you select one of the filter wheel Filter Wheel IMITA V Wheel A Shutter A options the interface will provide a set Wheel B Shutter B PER Eo of check boxes to tell iQ what you want Control Pot COME M Reconnect At Start to control as shown left Check the Speed 4 boxes of components you want to Shutter Delay fo ms Close Shutter On Move control Control Port l l The filter wheel is controlled through one of the serial ports on your computer From the Control Port dropdown select the port you are using From the Speed dropdown listbox select the Speed ha speed at which the filter wheel will change position Speed 1 is the fastest and speed 7 the slowest Note not all speeds are 1 2 3 4 5 6 f because filter load affects inertia and positioning performance The device has a self guaranteed to function without correction test utility for robust speed operation Refer to the manual for more information The Shutter Delay describes the time taken Shutter Delay 50 ke in milliseconds to open the shutter and to close the shutter Simply enter the value into the text box 465 Andor Technology Andor iQ USER GUIDE The minimum delay value is very important for continuous readout cameras for example video cameras and the Hamamatsu Orca 1 Specifying too low a value will result in the shutters opening and closing out of synch Tests at Andor with a Meteor controlled vide
5. V Auto Increment C nt M l Bleach On Selection Bleach Sample Device Position 103 0 105 0 10 ms NB if you Zoom the image please reselect Point tool 4 az Kk IV Automap 55 1363 445 494 Ss e Ed 5o 3 The MicroPoint device requires a minimum of 17 points for an adequate calibration When you are satisfied that you have sufficient points over the image then click on Next and the FRAPPA Calibration will be created by the system and used henceforth whenever a FRAPPA action is requested demanded 5 Setting the FRAPPA focus control Now that the FRAPPA system is calibrated in X and Y you can use it in FRAPPA on Demand mode This means you can go live and use a FRAPPA region type with a FRAPPA on Mouse Up action Place a FRAPPA region on the focused specimen image and right mouse on the region Select the FRAPPA on Mouse Up option when selected it will be checked Assuming the calibration above is accurate when you mouse up after moving the region the area immediately below the region in the specimen will be bleached Finally perform a series of FRAPPA tests within iQ making sure to test points and ROIS 143 O Andor Technology Andor iQ USER GUIDE 3 6 7 Mosaic Calibration The Mosaic Device must be installed and aligned by a fully trained Engineer 1 Preliminary setup requirements A we assume that you have an iQ Configuration that includes all the devices you wish
6. ad je SIMULATED_PSF_20001 tif e SIMULATED_PSF_20016 tif 3 Select the first file in the My Recent SIMULATED _PSF_20002 tif de SIMULATED PSF 200017 tif j Documents Me SIMULATED_PSF_20003 tiF fe SIMULATED PSF z0018 tif ke SIMULATED_PSF_20033 series in this case F SIMULATED PSF _z0004 tif SIMULATED _PSF_20019 tif SIMULATED_PSF_ 20034 je SIMULATED_PSF_20005 tif e SIMULATED_PSF_20020 tif Desktop SIMULATED _PSF_20006 tif SIMULATED _PSF_20071 tif S M U LAT E D PS F z0000 tif A je SIMULATED _PSF_20007 tif je SIMULATED_PSF_z0022 tif She SIMULATED PSF _z0037 j SIMULATEC PSF _z0008 tif SIMULATED_PSF_ 20023 tif SIMULATED PSF 20038 and click Open s Xe SIMULATED_PSF_20009 tiF e SIMULATED _PSF_z0024 tif SIMULATEC _PSF_z0010 tif SIMULATED PSF _z0025 tif je SIMULATED_PSF_20011 tif e SIMULATED _PSF_z0026 tif SIMULATED_PSF_20012 tif SIMULATED PSF 20027 tif je SIMULATED_PSF_20013 tiF Ae SIMULATED _PSF_z0028 tif SIMULATED _PSF_20014 tif SIMULATED PSF 20029 tif kS iili r nails File name Fw 4 The files will be opened as a eo a a Multi Load gt multi dimensional image in iQ Snap Photometics OnlineChart Princeton Instruments Ratio Imagen Ta F End 2 Userlet D End Visilog Zelgs LSM lem Andor SIF sif z MetaMorph Stack stk kl 5 If the files to be opened do not have a naming system outlined above but do have a common name it may be possible for them to be imported more
7. Chart Image Strip View 4D View 3D View Montage Kymography Orthogonal Slice View Selecting the View menu item provides the necessary tools for the adjustment of image contrast and brightness and selection of look up tables LUTs 5 1 1 LUT Control and Creation The LUT function is accessed from the View menu as described o above Clicking the right mouse button while the cursor is on the i Channel Icon below the image will also open up a pop up menu with a three entries LUT Mapping and Auto Map Mapping opens up a the Image Contrast Dialogue as described above The Auto Map Binary option has the same functionality as the Automap button discussed Select Colour above LUT opens up a selection of pre defined Palettes or Look ae Up Tables LUTs some of which are listed below Save LUT Grey In the Grey level Palette each image pixel grey value is represented by equal weights of red green and blue components Thus black grey level 0 has O O O White grey level 255 has 255 255 255 This is a standard method of visualizing monochrome images Negative The inverse of grey Palette where black appears white and vice versa Obtained by the 1 s complement of image grey levels Camera An input Palette from a camera where intensity is proportional to the video 238 Andor Technology Andor iQ USER GUIDE signal level i e this is a normal grey scale However grey level zero black is set to R
8. Gamma is the default mapping and generates a mapping of the form where Gamma has the range 0 1 to 8 0 This gives the user ultimate flexibility in enhancing images Gamma 1 Is linear 0 5 is square root and 2 0 is square Users can control the gamma coefficient value using the slider or the text box 243 Andor Technology Andor iQ USER GUIDE 5 2 The Online Graph The Chart Image is accessed from the View menu on the Image Window It will display intensity changes and or ratios in the Image Window both during Live Protocol execution and playback of previously captured data In short anything happening in the Image Window will be displayed by Chart Image It can use both data from ROls or the whole image Mean Channel Channel Name 3 i Seo N 6 sc FS i Darra H 4 fe pee E a eee Et j rs eo NA PA Soe Se er et S A tv tv es j e CEAO PETA REO 5 5 s Tt t Tt y i 5 m e gt 5 D v L eet Seah ia NA AO v C C f O 2 T Va MO a ee ee es Sos Ste eet eee e i 5 i 4_ feet do e p 4 E 5 AE PEA NN R 5 f 5 5 5 Ane 54 56 56 60 62 64 66 63 70 72 74 76 76 80 62 64 86 88 90 92 94 96 96 100
9. The File menu has two options Print and Close On completion of the experiment the charted data can be printed for further inspection and evaluation using the Print button Select the Close button to exits from the On Line Chart window 244 Andor Technology Andor iQ USER GUIDE The Channel Selection Menu opens the Chart Options Chart Options Channel Ratio Chart Options File Streaming COA Axis EE cea M During Acquisition qo During Animation Ok on A Ra ashes C Time Dimension l Separate Plots a Cancel I Show Legend Frame Number Frame Number M Show Event Labels Load All Regions Iv Enabled 50 Selected Region A L Scaling Channel Ratio fe Auto Scale fe Auto Scale C Manual Min fo Manual Min 0 Max 256 Max 3 The charted data can be presented in various formats dependent on the settings specified in the Chart Options Tab and whether user defined ROI s have been drawn in the image A discussion of IQ Analysis features in conjunction with the use of regions of interest is described in Section 5 5 3 Additional Tabs are available for specifying Chart Selections An overview of these options is described in Section 5 5 3 245 Andor Technology Andor iQ USER GUIDE 5 3 Through Series Analysis The Analysis Tab opens the Analysis Window which is used to calculate statistics for the image or for user defined r
10. When an image with Events is reviewed Navigator is shown You can see immediately how many events were created by the Events dropdown list box You can move to the frame where any event occurred by selecting the Event in the list as shown As you navigate through the image a text field is shown above the dropdown list as you pass each Event When you use the Analysis tab to perform through series analysis then the Markers or Events are shown on the data plot and you can also choose to show the Event labels This is illustrated over 234 Andor Technology Andor iQ USER GUIDE Acquisition Analysis AGE Analysis Calibration Movie Editor Spot Analysis The analysis plot can be printed ES and will appear as it does in this Graph Data Options blean dialog M AutoRange F Show Event sbels _ Pint Mean Intensity hai Picospritzer High paty Fura Ficospritzer High T 8 9 1011121314151617 18192021222324 25262729 4 8 4 Using the On Line Chart The On Line Chart allows you to monitor intensity PeT em in multiple ROIs as the protocol proceeds and Select item to insert images are acquired To insert an On Line Chart ie OLC you right click on the Protocol Tree on the age Du last Snap command and Select from the Insert Chane Ratio dialog right Note that you cannot use the OLC in Merge a Fast Time Tempus mode Protocol Cancel 235 Andor Technology Andor iQ USER GUIDE Th
11. Andor Technology Andor iQ USER GUIDE The second method uses a binary image usually created with a thresholding tool see next section from the original data set Then in areas of the binary image with value zero ratio processing is inhibited and the pixels set to the value specified by the user Creation of this binary mask image is handled through the mask wizard which is launched by clicking on the Create Mask Image button on the dialog shown above and is described in the next section 6 11 5 Create Mask Wizard Mask Wizard Setup Wizard When you create a mask image a Please select the Image Source wizard is launched and the dialog shown here appears At this point you select an image from the Me cl Image List to create the binary mask In this case the Fura2 image Source MINETSERVERADEMOIMAGESACA RATIOAFURA TIFI4 Stee is used In general this will be the Name FURA TIFI4 most common approach Then Type 12 bit grev pseudo Intensity 1 000000 lux 416 7 000000 pm y 242 1 000000 pm Wavelength 2o nm Time 200 ms click on the Next button Mask Wizard Setup Wizar Some pre processing can be applied to the image series before Please select the pre processing mode thresholding to create the mask When a time series contains cells that are static in the field of view Back Next gt gt Cancel we recommend use of the
12. Close Scan Summaries Current Scan Scan DemoScan S ieee Scan DemoScan ER Well Pattern 6 Well FS User Defined Fields Setup Channel Channel 1 2 3 4 Protocols Existing scans are displayed in the list at the bottom of the dialogue Clicking on the plus sign to the left of each item will display summary information about the scan To create a new scan press New A wizard will take you step by step through the creation of the scan displaying information to help the user at each step The first dialogue in the Wizard will ask you to select the type of scan The following options are available Protocols specify acquisition sequences An example screen shot from the Protocol Manager is illustrated below Protocols are created as prototypes basic forms and then edited as required by inserting and deleting elements Protocols persist between iQ sessions and are expressed as a Tree structure on the Acquisition page Section 2 6 1 The Tree structure is an active component which can be edited with right mouse pop up menus The prototype protocols in iQ 2 are always of type Time Lapse and are created with one Repeat Interval and a single channel named Current The user can edit protocols and insert nested Time XY and Z Repeat actions with Channels and other actions as desired The new iQ 2 Protocol structure makes iQ one of the most flexible acquisition platform available
13. Failed to Resize The Copied Remote Image ellipse is used to create a dual channel image based on matching the dimensionality of the image data selected on the right to that to that existing in the selected image on the left This means that you can construct a dual channel image from two single channel images 7 Create a merged image In the Constructor Edit box enter the click your chosen name for and The system will copy the file from merged image Constructor Master the Slave and create a merged image with your chosen name Show this image by selecting it in the Master List and clicking the Show button The example exhibits the expected vertical flip and has shown here been misaligned to emphasize the need for alignment Andor Camera With Fast Z and Wavelength File Edit Image List Region View FS Multi i wavelength Time Cho O Frame Number Min range Max range Increment Interval sims a gt fo fo f fi lt jooo fr ce ao SetMin Set Max esx se Automap 10002 367 132 5767 lt a id m i Oo o 8 Use Field Split to adjust alignment and rotation of the 2 channels Now select the Field Split Plug In from the Process Menu The merged image will be 122 Andor Technology Andor iQ USER GUIDE displayed in the Plug In If you Field Split Dual RG select the Merge button yo
14. Scan setup peo Flease enter a name forthe new Scan Qu er Microtitre 46 scan Options Mo BAulti vW ell Petr Scan C systematicall sampled tields User defined tlelds O Multifield Scan Ww Montage The Procedure for creating scans is shown in a convenient flow chart format in figure 4 3 1 on the next page The list of scan types supported in Andor iQ includes e Multi field or multi point user defined fields of view e Montage allows several fields to be scanned and stitched together to increase the field of view for larger specimens while maintaining resolution Montage patterns include Rectangle NxM fields Edge NxM fields are fitted to user defined feature edges boundaries and Linear a series of fields along a user defined line specified by fields at the ends of a feature e Multi well scanning is used for micro plate or multi slide patterns e g useful for multi treatment experiments Multi well allows capture of multiple 161 Andor Technology Andor iQ USER GUIDE fields per well using either systematic sampling or user defined fields e Multi field and Multi well can be extended with a Rectangle Montage at each field position e Multi field Montage and Multi well constitute the XY scans e XY scans can be combined with Z scans to study e g 3D development e XY scans can be enhanced using Autofocus to correct for stage drift over time e Autofocus settings must be recorded at each
15. 2 Now turn the 365 nm energy level down below the damage threshold and try the same thing with the MicroPoint pulsing continuously for several seconds if needed Make note of how long this takes again it will depend on the energy setting but also on the objective transmission and the focus of the MicroPoint 3 Now spot one of the filaments and trigger the MicroPoint to output 5 10 pulses You should see some bleaching if not increase the power a little until you do Now continue this process changing the focus in between bleaching actions reducing energy when possible at each step You should be able to get to the point of bleaching a single filament with a resultant spot in the order of the diffraction limit of the objective 4 Now when you go to the cell culture you may have to correct again by a similar approach to 3 above because you are now in water or thereabouts and focus will again be shifted Once you have setup MicroPoint for a given objective and specimen you should note the setting for later recall Andor Technology Andor iQ How To Documents A5 20 How to Create a Protocol Template Protocol templates provide an easy and convenient way for users to create protocols A template defines a basic skeleton that describes an acquisition patterns A template does not contain channel or scan information the user is responsible for adding channels and scan files It does however provide time repeat loops both n
16. 9 Protocol 1 P Protocol 2 41 Protocol 710 MW SS NoZDC 4 Protocol Basic mf test 4 Protocol bigmovespdtest 4 Protocol DualChT f3 Protocol FastTime 3 Protocol FastTime 4 af 3 Enter a name for the new Protocol E E r Necceescccscrecssesenses 4 Choose the Fast option if you plan to Use this option for FLZ functionality use the Protocol with Fast LZ or with Fast Time single Channel streaming H E 5 When you Click OK the Prototype Protocol will be generated and you can then Edit with the right mouse menu options 6 Right Mouse allows you to Insert Channels and Scans and Edit Repeat and Interval options Try the right mouse on all Protocol entries for a full range of options 535 Andor Technology Andor iQ How To Documents A5 5 How to Change the Sampling Rate on the Fly File Image List Settings QC Help Setup Wizard Protocols 1 When Protocol has been selected and the Run Protocol Manager button has been pressed the Protocol tree will appear under the Acquisition Tab in the Control Window New Delete Copy select Cancel Protocol Summaries 2 Make sure the Tree is expanded and Right Click on a e Interval In the picture the Interval is currently reading c FAST y Protocol Jen s Practice 001 500 ms nom th Protocol Onion Multichannel m Protocol r
17. Andor iQ USER GUIDE Snap is used to acquire 1 frame of an image On pressing Snap the label on the button will change to Stop To cancel Snap with a long exposure time press Stop to abort It Channel button shows the current selected Channel see screen shot 9 my E e Open Imaris Save Live Snap When you click on the button a popup Acquistion Analysis AGB Analysis Movie Editor Spot Analysis menu shows the list Status of Channels available and allows selection of one of these lf you select a Channel from the dropdown and then press Live iQ will drive the system hardware to the recorded Channel settings pre frame events associated with the channel will occur e g shutters will be opened and then Live video will be displayed in the Image Window 2 6 1 3 AutoSave Auto Save can be toggled on and off by clicking in the checkbox When selected the checkbox is labeled with a tick and on completing the current Protocol the image will automatically be saved to disk with the name Browse for Folder given in the Protocol and an extension including the selects Foder date and time Sg Local Disk Co Auto Save Bea CD RW Drive Di M C Kinetic igD ata Bi S users on elrond Us Se Filestore on elrond 2 ES e My Network Places H E iQ pictures To specify a folder in which to Auto Save press the button on the right A dialogue will ask you to select a folder
18. E Pered Te 20 tines OU Ad xx Device Setup H Scan MFx5F o E E Snap E Scan Test MF Compatibility End TO Loop Protocol i 4 i eee Hl Frappa Frappa FRAFFA All W Display Image Scan Z Scan Move Channel Ch l Show All Pepea T 200 times 0 me fastest ad gl Snap DE A End TO a Save As 4A Options 7 Help The dialog above shows a Protocol Tree as described in the previous section Scans can be INSERTED into this tree at the Repeat keyword by clicking the right mouse button and selecting Scan from the displayed list of Action keywords You then choose from the list of existing Scans To create anew Scan you must have a motorized XY and or Z device under iQ control On the Acquisition page press Scan to open the Scan Manager Press New to create a scan and the dialog left will show IMPORTANT ALL Multi well and Montage scans require that the system has been calibrated with Stage Alignment Wizard Section 3 2 Resulting scan patterns will not be correctly aligned and well sequencing may be incorrect if you fail to complete this step 160 Andor Technology Andor iQ USER GUIDE Please note that not all of the options above may be listed for your system The a E scan list depends on the Scan hardware devices available a a 2 kee Set Up New Scan and whether you have a license for multi field or multi well scanning N Next gt gt Cancel
19. If a boundary is set between two points it will be moved to the nearest point e lf other Periods require setup then either o Add another Period to the currently selected Point Position o Select another Point Position in the Point Data table and add the Period to the highlighted chart e Click Selected to perform the calculations for the selected Point Positions e Click All to perform the calculations for all of the Point Positions in the Point Data table e Once calculations have been made for any periods selecting them in the Point Data Table automatically populates the Period Data Table 5 13 5 Additional Analysis Options amp Features FRAP Analysis Protocol 246 File Mobility Chart Axis Mode Pre Bleach Correction From the menu the user may also e Zoom in and out on the graph by clicking and dragging along the X axis with the middle mouse button Click and drag right to zoom in click and drag left to Zoom out e Change the mobility calculation to either pure flow or pure diffusion e Change the x axis type The user can plot either from the point index or real time Note that any extrapolations are only shown when plotting in real time e Save data to file 338 Andor Technology Andor iQ USER GUIDE 5 13 6 Calculation Details Bt The pre bleach or reference region fit takes the form Ae Bt The recovery fit takes the form A l e The diffusion and flow quantities are determined using th
20. Mar ver E 1600 000 H Min channel image SA roo step Gamma Coefficient Ae FF Reh Ue Te ered RAS AEA Fe SP eas 1 00 m utomap Off V EU path rou SE Region of Interest Ok Cancel J 99 1 Contrast Wap oh mouse up Set the Max and Min mapping or scaling values in the image using the spin buttons or typing a number into the edit boxes on the right of the dialog Spin button step size is set by the Step setting Alternatively you can grab click and hold down the mouse the Blue Min and Green Max vertical bars on the histogram graphic and drag left or right The left mouse button controls the Min value while the right mouse 68 Andor Technology Andor iQ USER GUIDE button controls the Max value The image will be updated on the mouse up action To invert the mapping flip contrast check the 32 Image Contrast Options View Gamma m E 4955 000 Max Invert E 627 0000 Min Exclude Invert option To set values above Max to zero check the Exclude option To change the Gamma Coefficient at Oo SA ESR Geach oe OS rar ee TR CAME 1 00 Gamma z E 2859 000 H Max Invert i E 951 0000 Min Exclude Channel Mappings together select the Link Channels check box and use the Edit 0O boxes or graphical tools as described View Log Scale Histogram Zoom to Map Link Channels above and now both
21. bring up the same menus as discussed in Section 2 10 1 2 10 9 The Rectangle Button golos The Rectangle button allows you to define rectangular regions on the image for analysis They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and will be drawn in the corresponding color ROIs assigned to a feature are numbered sequentially in the order that they are drawn on the image Position the mouse where you want to place the top left corner of the rectangular region and holding down the left mouse button drag the mouse to the bottom right corner of the region and release the button Regions can be moved resized and deleted with the Hand button or the Pointer button Section 2 10 1 and 2 10 2 Also right clicking on a selected ROI will also bring up the same menus as discussed in Section 2 10 1 2 10 10 The Ellipse Button Ojo O 5 The Ellipse button will allow you to define ellipsoid regions on the image for analysis They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and will be drawn in the corresponding color ROI s assigned to a feature are numbered sequentially in the order that they are drawn on the image Position the mouse where you want to place the top left corner of the ellipsoid and holding down the left mouse button drag the mouse to the bottom right corner and release the button 97 Andor Technolo
22. contrast 128 Find what Find Next Direction Cancel Red Black Leyel 0 Red white Leyel 255 Up Down Green Black Leyel o L Match case Green white Leyel 255 Blue Black Level 0 Blue white Level 255 Integration control Bit L6 Integration State Bit l Integration Control Board 0 Integration Control Port Integration Camera Type 0 Mapping Min c Mapping Max 65535 Frame Interval hrs J 0 3 3 Search for the string DSD you may find more than one entry but you need to select the one entry which matches your current iQ Configuration 4 Change the Debug Options 0 to Debug Options 1 5 Save and Close the file 6 Open iQ with the same Configuration 7 Show the Device Setup dialog and when you switch to the DSD processing page you will see a series of tabs on the bottom of the dialog The DSD control tab allows you to Toggle Penrose on and off When you do this you will see the registration pattern and you will be able to gauge or measure whether it is centered by checking the location of its axis of symmetry see the figure below 8 The center of the CCD chip is at column 696 of the camera chip If the axis of symmetry is more than 20 pixels in either direction then you will need to adjust the relative position of the camera and DSD To do this feel the tension between the stage and the DSD C mount clamping screw and adjust and lock them in alternating steps while maintaining a center of symmetry close t
23. nDims are multi dimensional images first introduced in Kinetic Imaging s Lucida a legacy product for multi dimensional processing and analysis nDims are important because they are multi dimensional containers of the image data held on the lmageDisk and created when Protocols are run for acquisition or loaded from file The main advantage of an nDim is that it allows visualization processing analysis and storage of all image data as a single object Selecting and defining the dimensions is achieved with multi dimensional tools such as the Image List Section 2 5 2 Navigator Section 2 9 and Selector Section 2 8 2 6 All this combines to makes operations on the large data sets simple and rapid a real benefit to the user It also helps to minimize the difficulties associated with file management of multi dimensional datasets 37 Andor Technology Andor iQ USER GUIDE 2 3 8 Device Setup The Device Setup button on the Image and Control windows opens a dialogue for setting up and configuring all external hardware devices included with your system This dialogue is available to all Machine Users Only Local Administrators can change hardware settings e g COM ports board configurations etc the devices included different options will be Dependant upon available which are accessible from additional page tabs Information for most devices supported by iQ can be found in the manufacturers supplied manuals or i
24. see Average Average A common processing operation which is used to clean up data This is not a spatial average but one applied in the specified Dimension and Range of the selected n Dim So it can be used for temporal Z or Wavelength averaging Because averaging of this kind can either be Lumped or Running the user can select which mode of operation is preferred 374 Andor Technology Andor iQ USER GUIDE Range E g if you have a time series image of 500 frames and you wish to view the averaged data then you simply choose Time as the Processing Dimension The Range setting provides another level of control on the Processing Range Off the result will be a single frame which shows the average value of all pixels over the 500 frames Range Lumped the scroll bar will be activated and you can choose the number of frames over which you want averaging If you specify 10 frames for example then the result will be a 50 frame sequence in which each frame of the result is the average of 10 frames in the Source Result frame 1 R1 will be the average of source frame 1 10 S1 10 R2 will be the average of S11 20 and so on Range Running again the scroll bar will be activated and you can select the number of frames over which processing will operate If you specify 10 frames then this time the result will be a 490 frame sequence in which each frame of the result is the running average of
25. 12 ERAS selected region and 2 Qess214 8913 40360 46 7348 ee ee Peak Intensity the Peak intensity value of the selected region and time frame SD the Standard Deviation of the selected region and time frame Area the number of pixels within the region within the threshold limits specified in the Settings dialogue Section 5 4 1 250 Andor Technology Andor iQ USER GUIDE Area the percentage of the area within the threshold limits specified in the Settings dialogue Section 5 4 1 Integrated Intensity the integrated intensity within the threshold limits Specified in the Settings dialogue Section 5 4 1 lntegrated Intensity the percentage of the integration within the threshold limits specified in the Settings dialogue Section 5 4 1 Integrated Intensity Per Unit Area the integrated intensity divided by the number of pixels i e the area within the region Note That in the above diagram the displayed data is for Region 1 which is classified as Blue on the image to select a different color select the Pointer tool from the Image Toolbar right click on the region and select Classify lf Group Analysis is selected on the Options page the data is displayed for every Group color rather than every Region The Group column in the data table will list a second number in brackets e g 1 4 which signifies that there are four regions in that class 5 3 3 The Options Page Please refer to Section 5 5 for f
26. Andor Technology Andor iQ USER GUIDE The OK and Cancel functions can also be accessed from the menu bar at the top of the Data Review panel AES Plots Data OF Perimeter Cancel 1044 568 192 747 The Save Data button is used to save feature data to a results data file Any cells that have been marked for deletion will not be included The resulting data file can be read by standard spreadsheets such as Microsoft Excel Save Spot Data Save jr EA Kinetic J Kinetic _ SpotData a Goback PART F cil Network Neighborhood S My Briefcase A New Folder Save as type Spots Excel data file xld Cancel All the measured parameters specified previously for each feature are saved to the data file The Save Data and Clear Selection options are also accessible from the menu bar File Plats Cell No Save Data rimeter Cell 1 Clear Selection D FAF 291 Andor Technology Andor iQ USER GUIDE Feature selection for rejection is also used to determine which data to include for the plotting of histograms and scatter plots Binned histograms can be plotted for any of the available cell parameters For example plotting cell integrated intensity produces the typical Spot histogram The histogram plotting function is accessible via the Histogram button or from the Plots option on the Data Review menu bar Cell Histogram Perimeter 5Scatterplot 182 747 Similarly scatter plots to
27. Fields 32768 133 170000 Fields 32768 133 170000 Fields 32768 133 170000 Fields 32768 133 170000 Fields 32768 133 170000 Fields 32768 133 170000 Fields 32768 133 170000 Fields 32768 133 170000 Fields 32768 133 170000 18687 856224 15473 269787 19161 856224 15473 269787 19635 856224 15473 269787 21187 070280 14703 587234 21661 070280 14703 587234 21187 070280 15410 587234 21661 070280 15410 587234 22135 070280 15410 587234 8753 682553 17516 214056 9227 682553 17516 214056 8753 682553 18223 214056 9227 682553 18223 214056 9701 682553 18223 214056 11252 896609 17453 531503 11726 896609 17453 531503 11252 896609 18160 531503 11726 896609 18160 531503 12200 896609 18160 531503 Andor Technology Andor iQ USER GUIDE 912 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 116 500000 Fields 32768 116 500000 Fields 32768 116 500000 Fields 32768 116 500000 Fields 32768 137 330000 Fields 32768 137 330000 Fields 32768 137 330000 Fields 32768 137 330000 Fields 32768 141 500000 Fields 32768 137 330000 Fields 32768 137 330000 Fields 32768 137 330000 18750 538777 17265 483843 19224 538777 17265 483843 18750 538777 17972 483843 19224 538777 17972 483843 19698 538
28. Montage J Montage Positions 4 2 by 2 2 XY Positions 3 y Move XY 1035 5 755 6 28 85 2A Save As A Channel red A Channel green i Move Z 28 85 s a Options Channel blue 9 Repeat Channel CFP YFP 2 Help Channels Description vil See Se f The dialog above shows a Protocol Tree which is used to specify the sequence and timing of instrument controls functions including movements of stages excitation sources emission detector optics triggering and actions such as photo bleaching Channels define the system state required to create an image on the camera of a chosen fluorophore including illumination ports and techniques and usually will be set prior to a Snap action which opens shutters exposes the camera acquires the image data and closes the shutter Channels also contain image display information such as look up table LUT for pseudo color display and mapping max min range settings to ensure good image rendering This opens the Channel Manager where channels are set up and stored All existing channels are listed here for the current user Press the New button to continue 155 Andor Technology Andor iQ USER GUIDE Setup Wizar Enter a name for the new channel into the Create Channel text box New Channel The channel will be private to the current user unless the administrator is creating it lt lt Back Next gt gt Cancel
29. Time 200 me Approximate Save size calcium ion concentration with 80 5 MB time To process this image we must first select the ratio method one and then select the image from the Image List AINE TSERYERSDEMOIMAGE SSBIDAAD AND RELATED AN THER SO SLAMBDA TIF 17 by clicking the Select button 14 13 Lancel Help The dialog shown here will be displayed where images can be checked and selected Note that images must already be loaded into AQM Once selected the image name Fura2 is set into the Source Image box and a thumbnail of the image is shown on the graphic The Ratio Dimension Of Interest is set to Wavelength by clicking on the appropriate radio button The wavelengths for the ratio are input into the respective Numerator 340nm and Denominator 380nm text boxes using the two dropdown lists Then the background correction method can be selected as discussed below 6 11 3 Background Following definition of the ratio source and dimension of interest a background correction method must be specified for the numerator and denominator images Andor Technology Andor iQ USER GUIDE before proceeding to the next stage of the transformation set up Clicking on the Background tab reveals the panel displayed below ec Ratio eee File About Source Background Mask Calibration Numerator Backgroun Use constant value of i Use maximum of area of interest number Use i
30. checking the Establishing Communication Section at the bottom Device Resolution microns step of the Window If the values do not reflect those entered type the values again 483 Andor Technology Andor iQ USER GUIDE A3 7 3 The ASI Z Tab Enter the Device Resolution microns step Z The Default value is shown in the picture below Camera Lud Asi Stage Settings Device Resolution microns step Note The user must press Enter after changing the Z value Verify that the Value entered was set by checking the Establishing Communication Section at the bottom of the Window If the value does not reflect what was entered type the value again 484 Andor Technology Andor iQ USER GUIDE A3 8 Thor Labs LEDD1 controller Andor iQ can utilise the Thor Las LEDD1 LED light source controller via TTL I O from the Precision Control Unit PCU Generic Shutter device or the Kinetic Trigger device The LEDD1 is especially useful to control a white LED for rapid switching of bright field illumination The compatible LEDs are available housed in collimating housing with flanges suitable for all major microscope models as shown below The device has manual control as shown in CW mode the LED will be on and intensity controlled by knob rotation in Pulse mode the LED accepts TTL or analogue input The LEDD1 has a single input which can accept 0 5Volts DC and has a rise and fall time of lt 50 us If d
31. i Cathy2_confocal t0000 _20014 tif Cathy confocal t0000 20015 tif i Cathy2_confocal t0000 _z20016 tif i Cathy2 confocal t0000 _20017 tif Cathy _ confocal t0000 200186 tif Cathy confocal t0000 20019 tif i Cathy2_confocal t0000 _20020 tif Cathy _ confocal FOO00 20021 tif l Cathy confocal t0001 _ 20000 tif Cathy2 confocal togi 20001 tiF Cathy confocal t0001 20002 tif Cathy confocal t0001 20003 tif Cathy confocal t0001 20004 tif Ea Cathy confocal t0001 20005 tif i Cathy2_confocal_tO001_z0006 tiF Cathy2_confocal_t0000_20000 i Multi Load ha Initially selecting image red01 tif will subsequently Multi Load the Single frame formats Bitmaps Fenestra Kontron and TIFF 43 Andor Technology Andor iQ USER GUIDE Multi frame formats AVI usually compressed Biorad MRC ICS Microvoxel STK TIFF TIFF LZW A list of all supported file formats was discussed previously in Section 2 5 Note Bear in mind that if you try to save a multi dimensional image into a single plane image file then only the first frame will be saved Set Save Path specify the default directory to which experimental data is saved When you save experimental data the Save File dialogue will point to the specified directory by default Image Importer import an image or series of images from a 3 party program iQ uses its own naming convention x when importing or exporting
32. put a grid on the image Window see below and ask you to steer a high contrast feature into the top left region and then to the bottom right region By identifying the feature and reading the stage motion iQ can make an automatic calculation of the relative angle between the stage and the camera lt lt Back Next gt gt Cancel Stage Calibration 1 Use the stage to move itin xthen y Rotate the camera so that the feature aligns to the grid 2 Move the feature into the top left hand quadrant Click on the feature NB if you Zoom the image please reselect Point tool smo net of z y Current Fill Colour i Stage Position Reset Stage 25327 um 54978 um e 5A Atom vex era o ORO ole al 129 Andor Technology Andor iQ USER GUIDE The screen shot above shows the first stage of the angle measurement sequence A high contrast feature has been placed in the top left grid region using the stage joystick identified by a user mouse click and Point tool selected Next the feature will be navigated down to the bottom right grid region using the stage joystick A mouse click on the feature will allow iQ to capture its new position The screen shots are shown below lt lt Back Next gt gt Cancel Stage Calibration Using the stage move the original feature into the bottom right quadrant Click on the feature NB if you Zoom the image please resele
33. 1 1 Tab of the Control Window J Events a Pause Click OK to start protocol 25 Interval 1000 ms Protocol Tree is expanded in the Acquisition 2 Right Click End at the bottom of the O Repeat 100 times l 4 Channel Blue 1 Protocol Tree and the Insert button will b tap abbear Channel Green 2 PP os Sar End 9 Load Protocol 2 Channel 3 After selecting Insert the Insert Item Window will Insert Item appear Select Load Protocol from the List and press the Select item to insert OK button Press Cancel to exit at any time Move 4 After Selecting OK the Select Protocol window will open All available protocols will appear in the list Use the button to expand the details of the protocol Highlight the protocol you wish to use and Select Protocol press Select Press Cancel at any time to z elect the required Protocol I fa Protocol 2 Channel exit as Image 2 channel fF Protocol Type MC Keyboard Events On 27 Interval 88 me fastest Note The Protocol you want to run must be Repeat 50 times amp Channel Blue set up through the Protocol Wizard prior to Channel Green OnLineChart trying to insert and load into another protocol f facet Protocol tesing 5 Once selected Load Protocol along with the name of the Protocol will be added to the Protocol Tree under the Acquisition Tab see Seana example on the left celal 538 Andor Technology
34. 180 Andor Technology Andor iQ USER GUIDE which is accessed form the View menu in the Image Window The image above shows a blood vessel in a brain slice and was acquired with a Linear Montage in iQ ease 3 F Autoren a6 102 46 g This example shows the result of stitching a diagonal Linear Montage scan 181 Andor Technology Andor iQ USER GUIDE Y l acd E T Automep 70 1091255 146 x e x A A Montage reconstruction is shown above from one well of a micro plate We see cell nuclei labeled with Hoechst and F actin associated with focal adhesion protein Talin labeled with Rhodamine phalloidin Image supplied courtesy John Locke Karolinska Institute Sweden 182 Andor Technology Andor iQ USER GUIDE 4 3 6 Inserting Scans into Protocol Trees Once you have created XY Scans it is an easy matter to include them into a Frotocol Newtest oe Image Test Case 1 Frappa Multi Phase i Protocol Type Time Series ab Camera Binning Use Current Wait Allocation Off Camera Selection Use Current Events dP Pause Click OK to start protocol Move Channel Ch ior Delete 32 Sr ii End Channel i End TO Insert Item Select tem to insert Auto Focus o Move Channel Move Channel AND Snap Hint terate through Y 27 coordinate list 183 Protoc
35. Bright Objects he Lower Threshold 50 ak Upper Threshold 255 y Spot Analysis Optilas1 2Bit y Spot Analysis Optilas6Bit File Image List Process Function Wizards Plug Ins Help Setup Wizard Spot Analysis Set up analysis Bact Next gt gt Cancel Select Analysis Name 5 7 4 1 New Analysis Protocols A new experimental protocol is created by Analysis Name pressing the New button which reveals Select image to use in setup the panel shown right Select from Image List Before proceeding to the next page of the set up wizard you must enter the name of your file in the Analysis Name text box and select an image to be used for setting Ete cote Ceara l CATEMP COLONAT PIC up your experimental conditions C TEMP COLONAAVI_Colone CATEMP COLONA AY Multi Load CAOPTILAS SPOT SEQUENCE SBIT TIME_SHUT CAKINETICKTEST215 TIF An image can be selected from the Image dean zl List or captured from your current sample using the Snap option Once a file name has been specified and an image selected click the Next button to proceed to the next page of the wizard 279 Andor Technology Andor iQ USER GUIDE A panel is displayed to enable the specification of threshold limits for the analysis File Image List Process Function Wizards Plug Ins Help Setup Wizard Spot Analysis Set up analysis lt lt B
36. C Ext Trigger Timeout 1000 ms settings are locked and should only be Fastest Avail Memory MB 185 E Control modified by persons familiar with re amera Status i a C i ki camera settings See Section re al A3 2 2 2 2 for more information A3 2 2 2 1 The Exposure Tab Sub Window Sub indo Window 512 512 The Window button ven with a region defined on the image in the Image Window determines the specified X and Y dimensions of any image acquired when performing a Snap or running an Experiment Region Defining Tools on the Image Toolbar To specify the Region or Window simply select a Rectangular ROI from the Image Window Toolbar and drag it on the Live or Snapped Image You can right mouse edit this region to specify a size of exact requirements Then click on the Window button The camera will now be instructed to deliver only those pixels for each frame acquired This mode of operation can be used to speed acquisition substantially 449 Andor Technology Andor iQ USER GUIDE select Full Chip to revert the image back to the full chip size Note The Full Chip image size depends upon the Window Mode and Binning values selected Shutter Allows the user to Open and Close the shutter Shutter gt shutter or signifies that there is No f Open O Close Mo Internal Shutter Internal Shutter present Binning This is set by selecting from the list in the Binning dro
37. Channel Name Please enter a name tor the channel using the same Configuration EGFP in which case it will be available to all users It is recommended that the channel name should describe the fluorophore and or imaging mode where possible Press Next to continue through all connected devices This depends on your hardware configuration Set each device settings as desired 4 2 1 Important Note about shutters File ImageList Process Function Help SetupWizard Various devices have shutters e g filter wheels ALC CSU The state of the shutters must ensure that the desired Setup Sutter Filter Wheel for Orcal Sutter MC1 imaging path is open for illumination and lt lt Back Next gt gt Shutter2 Experiment Action Wheel Settings detection To minimize specimen exposure Current and the side effects of photo bleaching select the Open Close Option for the fastest shutter in the path Set slower shutters in the light path to the Open state and then they will not introduce unnecessary delays to every acquisition An example of a filter wheel Channel Wizard 7 Enable Wheel dialog with shutter set is shown left M Enable Shutter Shutter Open 156 Andor Technology Andor iQ USER GUIDE The Shutter Experiment Action state for shutters is only seen in the Channel Wizard hence all Channels must be created in the Wizard but can be edite
38. Command Status is located at the bottom of each tab This will signify the cameras or devices operating status The user can also press the Andor Icon at the bottom of the window to display the About Box Andor Blaimading formerly Kinetic Imaging Product il Establishing Communication ES ies a Version 6 03 29 T BuildTime 16 59 35 Feb 17 2005 i ee iain Copiright Andor Bioimaging Division 1993 Ax No Reply EE A Strelley Hall 100 Southcenter Court strelley Suite 900E 2 Nottingham Morrisville HGS EFE UK NC 27560 USA tel 44 175 906 1354 tel 6 1 919 467 DEDOS fax 44775 906 1353 fax 1919 467 0608 e mail Info kineticimading com e mail Info ustk ineticinading com WINK andoarig com The Lock option is only available to Local Machine Administrators LMA Pressing the Lock button gives the LMA a warning message Press OK to continue or Cancel to exit Select OK to lock or unlock the settings This is dependant on which mode the settings are in currently If the section is grayed out then it is locked If the section is not grayed out then it is not locked Once channels are set up or edited by the LMA the channel can be locked so no changes can be made by limited users Camera Ludi a Wheel A Settings Position Establishing Communication Hardware M Stag Control Filterwheel Fir 2r 3 4 M Zmotorcontrol Shutter TARD mA POS A Consult Your User Manual load Warning Chan
39. Protocol Tree Once the Protocol has been created iQ will switch back to the Acquisition panel where you can edit as desired Examples of Protocol editing are reviewed in the next section Existing Protocols can be selected by highlighting the name in the Protocol Manager list and pressing the Select button The Protocol Tree will be updated and from there it can be executed by clicking on the Run button Modifications to any protocol can be made by using the right mouse edit functions in the Protocol Tree the original 190 Andor Technology Andor iQ USER GUIDE protocol can only be overwritten by users with administrative rights Duplicate versions of any protocol can be created by highlighting the appropriate file name and pressing the Copy button Files can be erased from the list by highlighting the appropriate file name and pressing the Delete button Cancel exits from the Protocol Manager dialogue ignoring any changes Further details describing the creation of experimental protocols can be found in Section 4 8 4 4 2 The Flexible Protocol Tree are eer Andor iQ2 has a new look and new T Protocol es eg Image Test flexibility in the Protocol Tree The fF Protocol Type Time Series a af Camera Binning 2 2 Protocol Tree defines the acquisition Wait Allocation Off Camera Selection Use Current Action oe Events Sv Scan MFs3F Mon Montage Acquisition A typical Protocol Tree is shown lef
40. Wizard depends on the devices you have configured in your system so you need to extrapolate from the examples to your specific configuration Scans Scan Wizard provides tools for defining XY and Z scans ranging from simple multi point or multi field scans to micro plate or multi well patterns typically used in multi treatment screening experiments In addition the user can include a Z range with or without Autofocus and montage or mosaic patterns can be recorded around the field centers in cases where an extended field of view is required Protocols the Protocol wizard helps you set up and record the channels and scanning patterns repetition and timing of multi dimensional image acquisition sequences See sections 3 8 3 9 and 3 20 Andor Technology Andor iQ USER GUIDE 3 2 Calibrate Your System IMPORTANT Before you use Andor iQ to capture or analyze images you should calibrate the system If your system has a motorized stage then you should also complete Stage Alignment in order to ensure that scan patterns are accurate and in the correct orientation Careful attention to this process will help to yield the best results For multi camera systems and field splitting devices for simultaneous multi channel imaging calibration tools are accessed from the Wizards menus l0 x The stage has not been registered in this session of iQ This is required for correct scanning Scanning will not be avai
41. _ Nikon TE2000 Microscope Nikon TI Microscope Conix ZMotor Device Prior Filter Wheel Device _ precisExcite CoolLED _ Prior Stage Device L CRI MicroColor Device vi Sigma KOKI Device _ Leica DMR Device V Sutter Instruments Filter Device ll Nikon E1000 Device Sutter Instruments DG4 Device v Andor FRAPPA Device CITIL Monochromator Device Andor Generic Shutter Device Till Polychrome 5 Device MakKinetic Trigger Device _ Kinetic Stage Device v Yokogawa CSU Device Andor Technology Andor iQ USER GUIDE 4 1 1 Getting Started with Acquisition When you first start IQ2 if you have not transferred iQ1 X Protocols Channel and scans as outlined in section 2 3 1 4 then iQ2 will start up with a default Protocol as shown below In this case only the Channel Current will exist Current as the name suggests takes whatever settings are currently assigned to devices in the Device Setup dialog This default Protocol lets you start using iQ2 to acquire time series data right away You can adjust the camera and other device settings at will Acquisition Analysis Mowe Editor Spot Analysis Status e Auto Save C Andor iQD ata M Browne Revolution Time Lapse 4utolmag Image Protocol A Frotocol Type Time Series Camera Binning Use Current so A walt Allocation Off l Camera Selection Use Curent gee Events seed dP Pause Click OK to start pr
42. but different extensions e g Fast50 ChartOptions Fast50 KI_ EXP Fast50 Kl_ MERGE Fast50 KI_ NODE 550 Andor Technology Andor iQ How To Documents Fast50 KI_ RATIO To share the selected Protocol s with another user on a different system you simply copy all files with the same name and transport them to the other computer e g by email You can create a ZIP file using WinZip and send a ZIP package 4 Once the files are available on the second system then locate the equivalent Configuration folder remember this will be a Hidden Folder and simply copy all the files into that folder Note iQ MUST be Closed during this operation If you sent a Zip file the Extract operation may find the folder automatically if the Configurations on source and destination machines have the same name 5 When you next start iQ and select the appropriate Configuration you will the new Protocols will appear in the Protocol Manager 6 To Backup Protocols and Channels simply make a copy of the Configuration folder at our chosen location e g C Documents and Settings All Users Application Data Kinetic Imaging iQ Backup and then if you get a problem or a user makes undesirable changes you can revert the configuraiton folder to the Default setup form the back up folder 551 Andor Technology Andor iQ How To Documents A5 16 Setting FLZ Frame Transfer FT Blanking In FLZ mode we use TTL trigger signals form the PCU to keep the hard
43. evaluation computer Once the id is obtained we will issue an encrypted license file to run on that computer for a specified time period 1 3 3 Installation from CD or Download Insert the Andor Installation CD ROM and run the program Start exe from the root directory This will start the installation program which runs side by side with an installation Online Help Guide and will take you through the following steps 1 Andor Software Protection Key drivers for Windows 2000 Pro XP Pro Appendix 12 Andor Technology Andor iQ USER GUIDE 1 1 Plug in the dongle when instructed to do so by the installation program 2 Windows Scripting Host for Image Disk Configurator Appendix 1 5 3 Andor Bio imaging Software Appendix 1 6 4 Configuring your hardware devices Chapter 2 Appendix 2 and 3 5 Start using the software Section 1 4 Chapters 2 6 How To Documents For a detailed description of the installation procedure please refer to Appendix 1 If you need further help our technical support team will be glad to assist To report problems please go to http www andor com contact_us support_request Or if you prefer contact your local supplier who will then liaise with us directly If you are installing from a download then simply unzip the file you download and then run double click on the self extracting file that is unzipped with the extension exe Then follow on screen instructions The installation w
44. image enabling interactive setup of the camera The controls include Pre Amp Gain EM Gain and Exposure time Higher EM Gain can be used to reduce exposure time in low 157 Andor Technology Andor iQ USER GUIDE light level applications For some applications good image quality can be obtained by adjusting only the exposure time Higher EM Gain will reduce dynamic range but can completely overcome any read noise in the camera See Appendix 3 for further information regarding different camera setups The Channel settings include a Look Up Table LUT or palette which is applied to images acquired with that Channel s settings When contrast or Mapping Section 4 2 1 is adjusted Live in the Wizard the settings are Recorded in the Channel This ensures images are displayed with satisfactory contrast setup Wizard Channels Channel Manager New Edit Copy Delete Channel Summaries Current Channel EGFP Pye fe E 158 Channel red A Channel green Channel blue Channel EGFP A Camera EB Gain 0 AA Camera Binning CURRENT Camera Exposure Time fsi 0 04 fv LUT Green AA Sutter Filter vvheel 1 0 Sutter Shutter Current AA Sutter Filter vvheel 2 0 Sutter Shutter 2 Current e Channel Fura 2 340 nm Andor Technology Press Next to continue The Channel setup is now complete The new channel is listed in the Channel Manager a click on the sign di
45. is the gain automatically Pre Amp G ain 240 AD applied before electron multiplication Select a value from the dropdown list EM Gain Control Electron Multiplying EM Gain is the process in TE which one electrode is replaced with two FealEM Gain electrodes The first is held at a fixed potential Enables and the second is clocked as normal except that EZ gt much higher voltages are used than are necessary for charge transfer alone The large eea a electric field generated between the fixed voltage electrode and the clocked electrode is sufficiently high for the electrons to cause impact ionization as they transfer This is what causes the generation of new electrons and can increase camera sensitivity without increasing the exposure time The EMCCD gain may be applied to significantly improve the signal to noise ratio of weak signals enabling use of lower sample concentrations and shorter exposure 454 Andor Technology Andor iQ USER GUIDE times in high throughput screening approaches To set the EM Gain Control enter a number into the text box use the arrow keys to move up or down in increments of 1 or use the scroll bar Exposure Time This is specified either in seconds milliseconds Or EsposueTime M Frame Transfer microseconds The units are chosen from the drop down 300 ms 8 list while the value is set by the slider or typing into the
46. la PBC_VerticalFlip_Rotate160 16 Bit 642 0 1 Region Pye Fast MIP JU fa Pi AutorMap Image Background w Navigator w Center Image w Status Chart Image Strip view 4D view 3D view Montage kymography Orthogonal Slice view ToolBar toggles the display of the Image Toolbar Section 2 10 which is displayed above the Image Window Please note that some of these icons can be repositioned as necessary Bw NVA NN Tel DoS T img Navigator toggles the display of the Image Navigator Section 2 9 which is displayed below the Image Window Time z wavelength 0 00 00 000 himams Frame Number Min range Max range st o sia Set Min Set Max r Status toggles the display of the Image Window Status Bar which is displayed below the Image Window cal wa a Automap 100 21 205 85 O Andor Technology x ex E Andor iQ USER GUIDE 2 9 The Image Navigator The Image Navigator is displayed below the Image Window when Navigator is selected in the Image Window Tools Menu It is used to animate through multidimensional images and to select frames of interest for analysis Time wavelength Z 0 00 15 189 hmsms Frame Number Minrange bMaxrange o Increment o Interval sms a 23 o 199 i fotoa l a ra gt r me m ar Set Min Set Max Inject 1 Go to Event 1 Injecti 2 Inject 2 3 Inject 3 On top of the navigato
47. ms in the Unit semas Frish cma box and consequently 600 in the Tou need to input Time or dimension Identifier inthis page The Resolution box gives the more familiar identifier should be the text string that precedes the numerical index for the dimension h m s ms time format in the navigator Set Time toolbar Specifying s will give the time Time Identifier ne format in s only Unit Resolution ms Files FURA_time0000_wave0000 ti Press Finish The Multi Load progress bar will appear and the images will be loaded into the Image Window to be analyzed in the same way as a single multi dimensional image file 46 Andor Technology Andor iQ USER GUIDE 2 5 2 The Image List and Metadata Settings The Image List shows which images are held on the Image Disk All images are Opened or acquired and buffered on the Image Disk This provides rapid and efficient access to the large data sets associated with multi dimensional imaging Note Flexible protocol execution may create multiple image entries to support complex tree structures For more information see Chapter 4 Clicking on the Image List menu item opens the dialogue shown below o Image Manager C KineticlmageDisk Shared File Edit Comments Options Image Details Name RAT LIVER STACK TIF Path C DOCUMENTS AND SETTINGS M BROWNE DESKTOPSIO IMAGES DSD FIRST DATASOCT 20095 Type 14 bit grey pseudo Intensity 1 00
48. z Local Disk C z mageDisk Di s Curent Image Disk E se Data GB F Removable Disk G s s Images CE Bla Desktop My Documents a w oe 1394 A Camera PE C3 Shared Documents a CS My Documents My Computer d My Network Places Qu My Metwork Filename te Places Files of type TIFF Cancel 3 Select the file and click the Open button The image will appear in the iQ Image Window and also the ImageDisk 541 Andor Technology Andor iQ How To Documents A5 9 How to Import Multi dimensional Images Part 1 1 If the files to be opened have Andor iQ AE Image List Settings OC Wizards Analysis Process Image Edit Plug Ins Help a unique identifiable naming Open Image i i Save Image ee system for each dimension Set Save Path MEMUDEE ANDOR iC Settings Orca ER Ludi Piezo specifically a numerical ART BMP i i Image Importer system _2 to indicate Z ImageT ppe 8 bit grey pseudo 146 f Image Size 320 x 240 dimension _w to indicate a Display Range 1 to 146 wavelength or _t to indicate sheen RASAS m time select Open Image from the File menu in either the iQ control window or the image window 2 This will open the File Browser window Select the drive and folder in which the images are located By default iQ will look for TIFF files Select Multi Load from the dropdown menu Files of type Open File A SIMULATED PSF z0000 tif SIMULATED PSF z0015 tif
49. 1 Setup Print shows a plot for TN mean feature area Landscape for a time series comprising twenty time points MENEER EEE LULULULI UUTUBDU DO DO LULULUL Beset klaine M View Margins 0 L i UVAD UU DD CODO JLILULU LU 1 1 Clicking the Surface Plot radio button produces a 3 dimensional representation of the analysis data for the experimental parameter chosen previously in the Parameter Selection window The Surface Plot option can also be selected from the Plot menu Histogram of Area File Data Baia Animation All data Srimate Through Time z Statistical Plot Surface Plot 310 Andor Technology Andor iQ USER GUIDE An example plot is shown below feature areas measured for a twenty point time sequence Histogram of Area olx Fie Data Plot spots a oP POS Number of S i 5 3694 76 123 Time resolved data Global Main Peak F 504 ae 54 C All Data z ia el C Animate Through Time Min 504 504 Da 3781 F Animate Delays Ibs Bins 20 Frame 13 Mean 1051 655 Wee OSE Close Statistical plot SD 677 411 Update Display ize M tiean Mv so Cv 64 4 62 8 PESE Print h RR Mos m 1 Number rint gra Spots 102 57 SetDefaults Print aroph Surface Plot The orientation of the chart object in the display window can be adjusted by clicking on it using the left mouse button and dragging it to the
50. 10 frames in the Source Result frame 1 R1 will be the average of Source frame 1 10 S1 10 but R2 will be the average of S2 11 R2 A S3 12 and so on Differentiate Allows frame by frame subtraction in the chosen dimension At the most basic level in a time series you can produce a frame by frame difference and choose to subtract the first image from the second I i 1 I i or the second from the first I i I i 1 Maximum Projection This is a maximum intensity projection in which each pixel in the Result is maximum intensity pixel Same XY position in the dimension and Range selected Range works in the same manner as described above for Average E g This tool is useful for extended focus observation with Z series of fluorescent specimens or for highlighting feature tracks in T series There are many other uses Minimum Projection This is a minimum intensity projection in which each pixel in the Result is the minimum intensity pixel Same XY position in the dimension and Range selected Range works in the same manner as described above for Average This tool is useful for extended focus observation with Z series of bright field stained specimens or for highlighting feature tracks in T series There are many other uses 370 Andor Technology Andor iQ USER GUIDE Maximum Encoding This is an encoded projection in which each pixel in the Result is replaced by the dimension coordinate at which t
51. 101 Andor Technology Andor iQ USER GUIDE 3 1 Wizards Help Setting up Your Imaging System Andor iQ can be configured to control a wide range of hardware for many applications Not surprisingly we strongly recommend that you setup your hardware carefully in iQ before you use it for research imaging To help you we have created a number of Wizards for calibration of the system camera s and other devices such as microscopes motorized stages and photo bleaching and activation devices Many of these Wizards are outlined in this chapter and while we understand your desire to leap in it will serve you well to follow the relevant steps below to obtain the best performance from the software and imaging system The Wizards are accessed via the iQ 1 X Wizard menu and listed here together with the hardware they are designed to support and sections in this chapter in which they are described e Calibration Camera pixel microscope objective and C mount adapter basic requirement for real measurements in images See section 3 2 e Stage Registration iQ tries to send the stage to its coordinate origin this step is explained in section 3 2 and is used to ensure that iQ can reproduce stage positions from coordinates stored in previous sessions It is a basic tool to allow re use of scan patterns So if you try to cancel at start up iQ will Keep reminding you until it is completed for your current session e DSD Calibratio
52. 129 XY 2 19287 221 19764 698 129 XY 3 18813 221 20471 698 129 XY 4 19287 221 20471 698 129 XY 5 19761 221 20471 698 129 XY 1 21312 435 19702 015 124 83 XY 2 21786 435 19702 015 124 83 XY 3 21312 435 20409 015 124 83 XY 4 21786 435 20409 015 124 83 XY 5 22260 435 20409 015 124 83 XY 1 8879 048 22514 642 124 83 XY 2 93853 048 22514 642 124 83 XY 3 8879 048 23221 642 129 XY 4 9353 048 23221 642 129 XY 5 9827 048 23221 642 129 XY 1 11378 262 22451 96 120 67 XY 2 11852 262 22451 96 120 67 XY 3 11378 262 23158 96 145 66 XY 4 11852 262 23158 96 145 66 XY 5 12326 262 23158 96 120 67 XY 1 13877 476 22389 276 112 34 XY 2 14351 476 22389 276 112 34 XY 3 13877 476 23096 276 116 5 XY 4 14351 476 23096 276 112 34 XY 5 14825 476 23096 276 112 34 XY 1 16376 69 22326 595 108 17 XY 2 16850 69 22326 595 108 17 XY 3 16376 69 23033 595 108 17 XY 4 16850 69 23033 595 108 17 XY 5 17324 69 23033 595 108 17 Andor iQ USER GUIDE End Protocol Description End Grab Parameters Camera Andor Window Left 0 Window Top 0 Window Right 1003 919 End Well End XY Well 23 XY 1 18875 804 22263 912 104 XY 2 19349 904 22263 912 104 XY 3 18875 804 22970 912 104 XY 4 19349 904 22970 912 104 XY 5 19823 904 22970 912 104 Well 24 XY 1 21375 107 22201 229 137 33 XY 2 218
53. 140 120 100 80 60 40 20 40 60 80 100 120 140 160 180 20 Andor Technology 393 Andor iQ USER GUIDE 6 11 8 Comparison of two calibration methods We now present data from the HEK cell example showing the results of calcium ion concentration by the Grynkiewicz and Almers Neher methods Figure 6 11 1 HEK cells were loaded with Fura 2 and imaged with a Hamamatsu 4880 80 cooled CCD camera Kinetic Monokromator and Andor iQ The cells were treated with acetylcholine in three pulses and responded with elevated calcium levels Mean concentration data were obtained from the ROIs shown on the image Courtesy Dr Paul Thomas Department of Pharmacology Cambridge University Comparsion of Grynkiewicz and Almers Neher calibration methods in AQM Ratio Tool mean C1 G mean C2 G mean C3 G mean C1 A Ca2 nM mean C2 A mean C3 A 1 15 29 43 57 71 85 99 113 127 141 155 169 183 197 Time Figure 6 11 2 shows mean intensity vs time in HEK three cells Note heterogeneous response The concentration data produced by the two calibration methods cannot be distinguished on the graph above Tabulated data for 30 time points are shown in the table below Andor Technology Andor iQ USER GUIDE mean C1 G mean C2 G mean C3 G mean C1 A mean C2 A mean C3 A 39 0372 2 8282 34 1461 39 234 2 9689 34 3191 38 7552 2 9387 34 0805 38
54. 166 Andor Technology Andor iQ USER GUIDE Note Autofocus when you want to record and Autofocus setting at each field make sure that you have the Device Setup dialog visible and the microscope tab selected Then initialize the Autofocus button in the interface iQ should indicate if File Image List Settings QC Wizards Process Analysis Plug Ins Help the Autofocus was successful When Setup Wizard cane you click Next Field or Next Well iQ will Scan Manager record the current Autofocus setting from the microscope New Edit Delete Close Press Next Field and then repeat the Scan Summaries above to specify the positions of each Current Scan Large Areas field SETAS Scan Large Areas User Defined Fields 4 You will now return to the Scan Manager where the new scan has been added to the list Clicking on the plus sign to the left of the scan name displays summary Select Z Device information for the selected scan file 167 Andor Technology Andor iQ USER GUIDE 4 3 4 Multi well or Micro plate Scan pile Image List settings OC Wicards process Analysis Plug ins Help To start Multi well definition select the pone appropriate radio button in the Scan Set Up New Scan b Manager Then choose whether you want Systematic Sampling computer chosen or User defined fields to be Next gt gt imaged in the wells This will be Scan Setup
55. 19 rack mounted enclosure housing power supply and opto isolators with 8 input and 8 output BNC connectors for convenient coupling to other apparatus PCU provides a means of control and synchronization with external apparatus It will be especially useful for those wishing to synchronize with electro physiology equipment micro injectors flash lamps and other illumination control In addition the PCU box will allow coupling to third party apparatus that accept external TTL inputs to initiate actions The protocol described here is a physiological live cell experiment using neuronal cells It involves an initial period of depolarization of the cell and subsequent monitoring of intracellular calcium levels in response to stimulation with a calcium agonist e g acetylcholine The indicator used is the ratiometric calcium indicator Fura 2 In this example IQ is set up as follows two protocols are created for the calcium indicator Fura 2 which will be run sequentially by inserting a Load Protocol command Section 4 6 at the end of the first protocol named Fura pre drug 7 Protocol Fura pre diug Hg Image Fura ff Protocol Type MC A Trigger In Axon Start Sampling Start oa ate J A Events 3 Interval 88 me fastest Fai Repeat 50 times fF Channel Furas80nm 1 Scan Pd Snap Protacal Channel eg Channel Fura340nm 2 Device Setup l po Trigger Out Picospritzer Pulse W Di
56. 200 times O ms fastest Ao Move Channel Chi 2 Snap End 11 W End TO The FRAPPA protocol described above can be extended to provide repeated measurements of the process This could be achieved by repeated bleach recover actions on the same region or by moving the FRAPPA ROIl s around This is easy to achieve by introducing a new Repeat Time loop around all actions specified above as shown in the Tree below 4 6 3 Time Lapse Z Stacks with fast Burst at each Z This Protocol allows the capture of slow developmental events with a 5 minute sampling interval 5 hours observation in this case while also capturing fast bursts Py Protocol Test Case 5 Burst Within Z ES Image Test Case 5 Burst Within lt a Protocal Type Time Series a Camera Binning Use Current Wait Allocation Of Camera Selection Use Current Events dP Pause Click OK to start protocol gi Repeat T 60 times 5 min Ao Move Channel Ch Repeat 10 umin 11 planes a Snap ty End T1 jy End Z d End TO a Repeat T1 50 times O ms fastest 50 frames of physiological activity at different Z planes throughout the 5 hour process This kind of information is important in developmental biology applications where there are two time bases of interest to scientific study 211 Andor Technology Andor iQ USER GUIDE 4 6 4 Multi rate multi phase with Autofocus a Protocol Type Time Serie
57. 267 10 949 Cell14 4 833 000 144 000 42 000 0 505 51479 000 61 800 10 849 Cell15 4 729 000 122 000 37 000 0 615 9458 000 81 561 10 993 Cel 16 5 537 000 108 000 35 000 0 579 57259 000 106 628 10 955 m Celi7 A 554 NNN 11n NNN AR NNN n57 75758 NNN 136 747 11 235 se Total Spots 102 Histogram Scatterplot Save Data Clear Selection X Cancel Included Spots 102 All Data C Single Frame MEENA A File Plots Data View Perimeter Celt Jo 552 000 110 000 Cell 2 1 969 000 152 000 Cell 3 1 591 000 118 000 Cell 4 1 557 000 116 000 Cell 5 2 570 000 116 000 Cell E 2 534 000 116 000 Cell 7 2 683 000 144 000 Cell amp 3 506 000 116 000 The column Frame identifies the location of features analyzed in the temporal data series A data table can be displayed for the complete data set or a single time point by selecting the All Data or Single Frame option using the appropriate radio button The example above shows the complete data table for a twenty frame time sequence selection of data for the complete time series or a single time point can also be 305 Andor Technology Andor iQ USER GUIDE accomplished using the View option on the menu bar Data Review IO File Flos Data WHEY Frar All Data Perimeter Celi Oo aingle Frame 110 000 Cel2 1 568 000 152 000 Cel3 1 591 000 118 000 Selection of data corresponding to a single time point is illustrated below File Plots Data View Frame Area
58. 300 1 fo Y2 BB of the Field Split window Press the RETURN key to express changes You can also move the position of the two ROls by typing new x y values for the top left corner of the ROIs into the Edit Box named X1 Y1 X2 Y2 then press the RETURN key to express changes You can have x or y profile across the two ROIs displayed by checking pum the check boxes named Profile X and Profile Y to help you judge the wi Pcie result of the alignment The profiles displayed have blue and yellow colors for ROI1 and ROI2 to distinguish between the two ROls The profiles are from a point marked by a small blue or yellow cross within the ROI you can move the point by using mouse to drag and drop or by typing new values into the Edit Boxes named Xp and Yp at the bottom 350 Andor Technology Andor iQ USER GUIDE of the Field Split window ROI1 and ROI2 have already been assigned Channel 1 Channel2 650 nmi 550 nm wavelengths and LUTs that are listed at the Red Green right bottom of the Plug In window You can change the wave length values or assign new LUTs to them as necessary aK Once you are happy with the two ROls and the new double channel image and then click the OK button on top right of the Field Split window This will generate a new image on the ImageDisk and have it displayed in the Image Window as shown in the figure below and will close the Field Split win
59. 34 Andor Technology Andor iQ USER GUIDE File Image List Settings QC Wizards Plug Ins Help More advanced Protocols can Setup Wizard Protocols include multiple channels scans Protocol Manager moves trigger in out user defined delays on line processing on line graphing REW Delete Copy select Cancel pauses which require user input Protocol Summaries Current Protocol Furae Se di Frotocal Fu raz B Image Fura a Protocol Type MC keyboard Events On SS Interval 88 ms fastest a Repeat 50 times of Channel Fura380nm Please refer to Section 4 5 and 4 6 for a description on how to create and use protocols 2 3 5 Triggers and Events Andor iQ now has a powerful set of Trigger commands for protocol control External synchronization of imaging systems with other forms of data acquisition specimen stimulation or specimen treatment is important in various scenarios Triggers are grouped as Trigger IN and Trigger OUT commands for inclusion in the acquisition protocol from the Right mouse pop up menu Triggers are given simple text names to ease user interpretation through the Trigger manager and are used to monitor and control 8 inputs and 8 outputs Each of these input outputs is associated with a TTL level signal linked into the imaging system through Andor s PCU or BOB Trigger Boxes Each signal is coupled through a BNC connector on the Trigger
60. 3Ch MF Protocol Type Time Series Camera Binning Use Current Wait Allocation Off Camera Selection Use Current Events Keyboard Events On Default name Spacebar Scan Multi Well 6x4 5SysSampling Pause Click OK to start protocol Interval O ms fastest Repeat 1 times Repeat Well Repeat XY Sampling Density 5 Scan Pattern 6x4 2mm 2 5mm plate 515 Andor Technology Andor iQ USER GUIDE 516 Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Andor Technology XY 1 8691 15017 124 83 XY 2 9165 1501 7 124 83 XY 3 8691 15724 124 83 XY 4 9165 15724 124 83 XY 5 9639 15724 124 83 XY 1 11190 214 14954 316 124 83 XY 2 11664 214 14954 316 95 67 XY 3 11190 214 15661 316 95 67 XY 4 11664 214 15661 316 95 67 XY 5 12138 214 15661 316 95 67 XY 1 13689 428 14891 635 120 67 XY 2 14163 428 14891 635 120 67 XY 3 13689 428 15598 635 120 67 XY 4 14163 428 15598 635 120 67 XY 5 14637 428 15598 635 120 67 XY 1 16188 642 14828 951 104 XY 2 16662 642 14828 951 116 5 XY 3 16188 642 15535 851 120 67 XY 4 16662 642 15535 851 119 31 XY 5 17136 642 15535 851 87 34 XY 1 18687 856 14766 27 116 5 XY 2 19161 856 14766 27 116 5 XY 3 18687 856 15473 27 116 5 XY 4 19161 856 15473 27 116 5 XY 5 19635 856 15473 27 116 5 XY 1 21187 07 14703 587 112 34 XY 2 21661 07 14703 587 112 34 XY 3
61. 5 Gnd Channel 2 Switch Input Pin 11 Pin 14 Channel 4 Switch Input Pin 13 Pin 14 Trigger TTL Input Pin9 ve Pin 2 Gnd Channel 4 TTL Input Pin13 ve Pin 6 Gnd Channel 3 Switch Input Pin12 Pin 14 Sync TTL Output Pin 15 ve Pin 7 Gnd IPin 1 8 Gnd Connecting a switch contacts between the switch input pin and the internal 5V pin14 will turn on that channel when the switch is closed Pin1 8 Gnd TTL Source TTL Sync CoolLED 15 way FiesMB 9 Trigger FireSMB Gnd Te to Channel 1 O Gnd 8G Tm tt Channel 2 engine ms 2 channels engine ma ia channel O engine Tine oat 15 Syne TT O Gnd 7 Gnd 490 Andor Technology Andor iQ USER GUIDE A3 10 Andor Differential Spinning Disk DSD The Andor DSD scanner enables confocal imaging combining the advantages of a spinning disk with structured illumination iQ controls allow the DSD to switch internal filter wheels for excitation and emission internal mask for image calibration and Spinning disk between its high sectioning and high signal positions The interface to the DSD is via USB and the actions required for calibration and background corrections are handled automatically and so are not covered here but in Chapter 3 of the user guide The Andor DSD is shown below with its replaceable filter turret In normal operation the cover will be in place and the turret installed Details of filter turret replacement can be found in the iQ Wizards menu User a
62. 650 Wavelength nm 35 UV Blue Cyan Teal Green Yellow Red 30 25 20 Relative Spectral Power mW nm 4 A 400 LED light pumps enable high speed modulation currently 5 kHz with rise and fall times of lt 10 us with typical power stability lt 1 of peak Since LEDs are extinguished whenever a channel is OFF there is no leakage of UV light which can be highly damaging to live cell specimens LLE has a typical lifetime of 10k hours ensuring a low cost of ownership The source geometry leads to highest brightness at the center of the luminescent crystal core and when coupled into a 1mm 0 22 NA silica fiber LLE delivers up to 5 X power into a given etendue than a conventional arc lamp e g metal halide The combination of these qualities makes the LLE a desirable source for many applications in fluorescence microscopy where specimen longevity and data quality are key factors You can control the LLE in iQ with a simple configuration of either the Generic 572 Andor Technology 573 Andor iQ How To Documents Shutter or the Trigger and Event device controller The sequence that follows shows how to setup the Generic Shutter and how to configure for use with Andor s DSD but the average user should be able to extrapolate this to his her specific setup 1 Build a Configuration or add to your existing configuration the Generic Shutter device adapter when you work with
63. 9485 28 0773 34 2483 38 9572 27 4591 34 1447 39 1502 27 5971 34 3167 39 2263 2 8933 34 3041 39 4218 28 0332 34 4778 38 7477 27 4831 33 5238 38 9421 2 6226 33 6957 39 6307 27 4859 33 9895 39 8273 2 6212 34 1576 39 0977 27 1118 33 455 39 2952 27 2462 33 6222 39 6248 2 685 33 6357 39 8265 2 8252 33 8056 40 6566 28 3887 34 6258 40 8607 28 5306 34 7968 40 1485 2 524 33 5718 40 3522 2 6613 33 7404 40 0178 2 9969 34 1665 40 2173 28 1338 34 3354 39 5892 28 2121 33 9673 39 7866 28 3521 34 1343 40 8451 29 1231 35 2416 41 0491 29 2719 35 4202 38 7934 2 4939 33 6023 38 9926 2 6316 33 7729 39 4562 28 3069 33 8227 39 6543 28 4485 33 9925 38 7657 28 1186 33 3381 38 9635 28 2625 33 5061 40 1197 29 5165 34 8588 40 3222 29 6638 35 0337 39 5347 28 0738 33 647 39 7385 28 2153 33 8171 40 6812 29 0011 34 5001 40 8833 29 1471 34 6741 40 372 28 501 34 2063 40 5756 28 6439 34 3783 40 2071 28 5905 33 9559 40 4086 28 7355 34 1275 189 6756 30 9373 43 1931 190 7208 31 0923 43 4127 350 1005 71 46 356 1057 352 2077 71 8353 358 2575 252 381 71 4903 376 9319 254 1812 77 9009 379 2376 194 1308 65 0202 245 6351 195 2011 65 3585 247 0312 178 5373 55 7029 169 4519 179 5111 55 8906 170 374 190 5858 49 7103 129 3414 191 6335 49 9623 130 0298 212 0788 44 9895 108 3797 213 2619 45 2194 108 9489 217 1133 44 5086 98 8777 218 3241 44 1374 99 3896 211 0195 43 005 92 5054 212 1955 43 2229 92 9903 Table shows a comparison of Ca2 concentration 30 time points after calibration
64. Andor iQ How To Documents A5 7 How to On Line Chart During Acquisition 1 Make sure Regions of Interest are selected on the image Select View from the Image Window and select Online Graph File Edit ImageList Region Fast MIP Jo x ajaja d kimp e T x Fo Toolbars Image Background Center Image Chart Image Strip View 4D view 3D view Montage Kymography Orthogonal Slice View 2 The Mean Chart window will appear You can re position and resize this window like any other 3 If you have regions selected on the current image in the Image Window from a Protocol or animating under Navigator control you will see the Chart update with the image updates Chart computes means of ROI s AND Mean of Ratios of pixels in the ROls in dual channel Protocols J x UNE Ratio Channel 54 56 56 60 62 64 66 66 70 72 76 50 62 86 90 92 94 96 98 100 539 Andor Technology Andor iQ How To Documents Channel Selection 4 To Print or Close select File from the Menu Bar 5 To modify the Chart Settings select Channel Selection from the Menu Bar Make selections for all tabs and press Close Chart Options Channel Ratio Chart Options Chane cI W euen Solid Cos B Green 2 Solid Note The Acquisition will pause Chart Options ey ec when selecting Chart or changing Rata amaeaaaeaeamamaeaeaaeasssssOOIIIIOS i 1 me cose Settings and will resume when Ta zi Clos
65. Area JES Data Plot Close 5 7 6 4 Scatter Plots and Editing Scatter plots can be plotted for the comparison of any pair of feature parameters by clicking the Scatterplot button or accessing the function from the menu bar as described previously Parameters to be plotted are selected by clicking on the appropriate radio buttons in the Parameter Selection window The resultant scatter plot is displayed after Select Parameters for scatterPlo op oete ee clicking the OK button 6 Area O Area Perimeter Perimeter C Max Chard C Max Chard C Shape C Shape C Integrated Intensity Integrated Intensity C Mean Intensity C Mean Intensity C Log Int Intensity C Log Int Intensity C Mean Int Area C Mean Int f Area Cancel An example scatter plot of Area Vs Integrated Intensity is shown below 299 Andor Technology Andor iQ USER GUIDE Scatterplot of Area and Integrated Intensity In the upper part of the dialogue box a Scatter Plot of the chosen cell parameters is plotted Clicking on the Cancel button exits the dialogue and returns to the Data Review panel The Select Area button enables the user to select an area or multiple areas from the scatter plot If areas have been defined on the plot any points not enclosed in these areas will be checked for deletion on exiting the Scatter Plot dialogue with the OK button The OK and Cancel functions are also accessible from the menu bar
66. Autofocus setting at each field make sure that you have the Device Setup dialog visible and the microscope tab selected Then click on the Initialize the Autofocus button in the GUI for each field 174 Andor Technology Andor iQ USER GUIDE required iQ should indicate if the Autofocus was successful When you click Next Field or Next Well iQ will record the Autofocus setting from the microscope Press Next at the top of the field definition dialog to continue You will now return to the Scan Manager where the new scan has been added to the list Clicking on the plus sign to the left of the scan name displays summary information about the scan File Image List Process Function Wizards Plug Ins Help setup Vizard cans Scan Manager Mew Edit Delete can Summaries Current Scan MAW y S S CH 1 o Calibration x40 objective maa Setup Channel CH1 175 Andor Technology Andor iQ USER GUIDE 4 3 5 Montage scans We can use Montage scanning in either a single scan definition or as part of a multi field or multi well scan In the multi field and multi well cases montage is constrained to be a rectangular region specified in mm or fields centered on each field of those scans The montage fields are automatically included with each field when you select Montage with Multi Field or Multi well in the Scan Manager You choose how large the montage at each field will be Note BEFORE you define
67. EM gain is applied Section A3 2 2 2 Gains In this mode the camera provides the lowest readout noise If the EM gain and EM amplifier are used then readout noise will be higher but in very low light conditions EM gain amplifies detected signal far above the readout noise In the 887B ECS model with conventional readout mode at 1MHz Readout the ADC can be switched to channel 1 which provides 16 bit grey resolution and maximum dynamic range Vertical Clock Voltage Vertical Shift soeed and voltage of the CCD can be controlled to optimize charge transfer and minimize spurious background EMCCD imaging sensors exhibit some unique features At the present time all EMCCD sensors are of Frame Transfer architecture and require a vertical shift of the image into a storage CCD register prior to read out The speed and voltage applied during this vertical shift significantly affect the quality of the image and the background noise events It has been observed that a faster vertical shift leads to lower background or spurious charge events and therefore lower noise Faster shift can also lead to lower charge transfer efficiency which may result in a residual ghost image on the image area after readout To overcome this effect the Vertical shift voltage can be increased 1 2 3 or 4 456 Andor Technology Andor iQ USER GUIDE In our experience the best default settings for fluorescence imaging are a Vertical shift Time of 1 8 us and set
68. Ellipse Polygon Freehand Polygon Text Stamp Scale Bar Scale Line Copy Copy the selected region Paste Pastes a region that has been selected with the Copy command The pasted region will be identical in size shape and class to the original region It will overlay the original region but can be moved using the Pointer button on the Image Toolbar Delete Deletes the selected region from the image Delete All Deletes all regions from the image 91 Andor Technology Andor iQ USER GUIDE Classify Assign a color to the selected region This is relevant when performing a Group Analysis Section 4 6 in which results are calculated for all regions on the image in the same class i e for all regions of the same color as opposed to being calculated for each individual region Also with Group Analysis regions in the same class will exclude overlapping areas from analysis Regions can be drawn within regions to create holes and regions within holes to create islands Load Save The Feature Definition list Section 2 8 4 can be saved to disk from where it can be reloaded together with the position and class of all ROI s defined on the image Move To and Set Reference If a motorized XY stage is currently under iQ control then images acquired with iQ have stage coordinates attached in the Metadata Using this information and providing the stage has been registered or sent home with the Stage Regist
69. Exit Software Protection Troubleshooter Read Your Software Protection Key Read Done Version Level Get Donale ID For Kinetic Customers who are Upgrading Existing Software Installing A Second Kinetic Product or g Activating Kinetic Plugins contact Kinetic with this Dongle ID number and you will be supplied with an initailisation code Initialise Dongle 1039623 1 34273 32091 9 624891 879934 Press the Initialise Dongle Button Exit Andor Configuration Manager 424 Andor Technology Andor iQ USER GUIDE A1 4 Install the Latest Windows NT Service Pack If running under Windows NT we recommend installing the latest Windows Service Pack which is included on the Andor Installation CD Rom 1 If the installation program is not already running insert your Andor Installation CD Rom into your CD Rom drive and run the program Start exe which is in the root directory of the CD This will start the installation program which runs side by side with an installation Online Help Guide 2 Select the Help and Utilities page and click on the text Install Windows NT Service Pack 3 Follow the on screen prompts accepting the default values The Service Pack will be installed on your system after which you will need to reboot your computer Note You can also install the Windows NT Service Pack from you CD Rom without Starting the installation program From the 1386 Update folder on the CD Ro
70. Frame Action Output DO1 Trigger Out 0 MPUDOU Low Trigger Out 1 MPUDO1 High Trigger Out 2 Current Trigger Out 3 Current Trigger Out 4 Current Trigger Out 5 Current Trigger Out 6 Current Trigger Out 7 Current 486 Andor Technology Andor iQ USER GUIDE A3 8 2 Note on optimizing transmitted light image quality When using LEDD1 and other LEDs for transmitted light imaging with the CSU laser dual spinning disk confocal you may be subject to scan line artefacts in resulting images In order to minimize this effect you can introduce a neutral density filter NDF into the transmitted light path so that longer integration times can be used without saturating the camera 487 Andor Technology Andor iQ USER GUIDE A3 9 CooLED PE2 and PE100 LED illuminators Andor iQ controls the CoolLED PrecisExcite PE2 and PE100 LED The PE devices PE2 illustrated here are available from Andor Technology and integrate with Andor systems and cameras PE2 is available with 2 3 or 4 LED wavelengths The LED array modules LAMs and can be switched by the user for increased flexibility A3 9 1 Using PE2 with Andor iQ OAA YEE You control the CoolLED PrecisExcite LED m z tve usos B nes o PEZ from iQ via a USB interface which is Camera CoolLED mapped to a COM port usually Port 9 Control Thread Delay Settings LED Selection When you have built a Configuration using T the P
71. IDE ATAZATAPI controllers Fie Keyboards H T Mice and other pointing devices H Monitors Ee Network adapters o Other devices _ PCI Modem eg PCMCIA adapters El Ports COM amp LPT oo Communications Port COM 2 ba Y ti Printer Port LPT 1 H Processors H Sound video and game controllers 23 System devices F Universal Serial Bus controllers gt The Parallel port should appear as ECP or EPP in Device Manager If it does not appear or is listed as SPP restart the PC and change the parallel port settings in the BIOS If you are not happy with this step contact IT support or a computer competent person 423 Andor Technology Andor iQ USER GUIDE A1 3 Initialising a Dongle for Additional Products If you have purchased an Andor Product and Dongle and now wish to install and run another the Dongle will need to be updated to allow the new software to run This is performed within Configuration Manager Section 2 3 1 and Appendix 2 When an additional product is purchased Andor ClearView for example a dongle initialisation code will be supplied Run the Configuration Manager from Start gt Programs gt Andor Bio imaging Click the Settings menu item and select Software Protection Key Input the initialisation code including hyphens into the blank space to the right of the Initialise Dongle Button te Kinetic Device Configuration Manager 7 X Fie Edit Settings Tools About
72. LastSession KI_MERGE 1 KBE KI_MERGE File 12 13 2010 9 51 AM CI ikon Prior 1X81 Piezo ALC Frappa a LastSession KI_LZEXP 7 KB KI_LZEXP File 12 13 2010 9 51 AM oe iXon TI ALC Piezo LastSession KI_ExP 3KB KI_EXP File 12 13 2010 9 51 AM No Grab 2 LastSession ChartOptions 1KB CHARTOPTI 12 13 2010 9 51 AM Jw Memory Leak Test KI RATIO 1KB KILRATIOFile 12 13 20109 254M 7 99 KB Type KI EXP File Date Modified 12 13 2010 9 46 AM Size 7 99 KB My Computer Andor Technology 570 Andor iQ How To Documents Using explorer navigate to C Program Files Andor Bioimaging Common Files Templates or the equivalent for your installation of iQ Paste the file Templates File Edit wiew Favorites Tools Help Q sax S iF search E Folders IFS es es id m Address lo G Program Files Andor Bioimaging Common Files Templates E Go E 3 Program Files 2KB KI_EXP File 1 O Acronis MCZ 2 First KI_EXP 2KB KI_EXP File 1 AGEIA Technologies gt MCZ KI EXP 2KB KI_EXP File 1 E Andor Bioimaging MultiPhase KI_EXP 3KB KI_EXP File j 2 2Alpha MultiPhasewithFrappa KI EXP 3KB KI_EXP File i Backup For iQ2 Testing 23 07 10 Common Files 5 MultiRate KI_ExP 2KB KI_EXP File 1 32 Backup of Common Files for Biophysical a Ratio KI_EXP 2KB KI_EXP File El B Common Files 5 MyTemplate KI_ EXP SKB KI EXPFile O ConfigurationFilearchive O DSDProcessing O Graphics
73. Number Of Fields 9 3 by 3 umber Of Fields 9 3 by 3 automatically Field Overlap 5 3 177 Andor Technology Andor iQ USER GUIDE Setup XYZ options Select Z Device Dewice Setup lt lt Back Next gt gt Montage Region Definition Steer the stage to locate an edge or centre position Press the appropriate registration button x position 63467 y position 28506 z position 0 gi oom Z Position ae eT ee S ae s 0 gt gt gt gt gt gt In Region mode you define the size of the region at the first step and then with the wizard panel shown here you can adjust the stage and when you click on one of the 9 3x3 buttons you specify the current field of view to be at that location within the Montage E g if you click to top right button the field becomes the top right field of the Montage and if you click the center button then it becomes the central field and so on When you define the Montage scan the stage graphic will be shown as below to provide a context and visual feedback Large Areas 16 200 16 300 16 400 16 500 16 600 16 700 15 600 15 600 16 000 16 200 Montage Current Position Current Position 15 x 16 400 16 600 178 Andor Technology Andor iQ USER GUIDE Andor iQ 1 10 1 Andor Camera 5 x File Image List Settings OC Wizards Process Analysis Plug Ins Help Setup Wizard setup XYZ options URSS
74. Perimeter Max Chord Shape Int Int Mean Int Log Int Int Cell 2 1 868 000 152 000 49 000 0 472 119806 000 138 025 11 694 Cell 3 1 591 000 118 000 36 000 0 533 94156 000 159 316 11 453 Cell 4 1 557 000 116 000 35 000 0 520 89870 000 161 346 11 406 4 El Total Spots 3 Histogram Scatterplot Save Data Clear Selection Jf OK X Cancel Included Spots 3 C All Data Current Frame 1 Single Frame E E Image Review OF XI Data for the required frame is viewed by S2beFtctsssed Image to Review CA TEMP COLONA AVI Colonal adjustment of the slider bar The functionality of the Histogram Scatierplot Save Data Clear Selection OK and Cancel buttons has been described previously in Section 5 7 6 1 CurrentFrame 0 0 j E 19 View Close For a data set generated from a time series the Show Images Image Review window appears as follows Individual features with their superimposed bounding rectangles and identification numbers can be visualized for any point in the time sequence by selecting the 306 Andor Technology Andor iQ USER GUIDE appropriate frame using the slider bar and clicking the View button The Close button exits from the Show Images dialogue 5 7 7 1 Dynamic Histograms Additional functionality for the analysis of temporal data is provided within the Histogram plotting function Histogram of Area EST x File Data Plot To plot a histogram click on the Histo
75. Repeats and Interval When the 198 Andor Technology Andor iQ USER GUIDE Protocol is running if you right mouse on the Action you can Edit the interval but not the number of repeats If you use Interval Setup under the Settings menu then you can create a predefined list of intervals from which you can select during Protocol execution Return to Table Repeat Montage Iterate through a series of Montage fields This Action is implicit within the XY Scan settings it is not inserted independently If this Action is deleted the entire XY Scan will be removed from the Tree If you choose to Edit this item it is possible to Edit the entire XY Scan Return to Table Repeat Well lterate through a series of Wells in a multi well or microtitre plate This Action is implicit within the XY Scan settings it is not inserted independently If this Action is deleted the entire XY Scan will be removed from the Tree If you choose to Edit this item it is possible to Edit the entire XY Scan Return to Table Repeat XY Iterate through a series of XY fields with optional Montage and Well iterators if defined in the XY Scan In the Insert Item dialog you select an XY Scan Note that you can insert only ONE XY Scan in the iQ Protocol Tree The field coordinates will be absolute if the Stage Registration wizard has been run You will have a common zero position between sessions and coordinates will be re usable The XY and Z coordinates of the fie
76. Scale CO Manual Min C Manual din Ma Mas 3 Select Close to accept any changes and exit the chart Options window Settings can be recorded for future use with Load or Save buttons on the right hand side of the Chart Options Tab 206 Andor Technology Andor iQ USER GUIDE 4 5 3 A Multi Channel Protocol with Z Scan File Image List Settings Wizards Plug Ins Help Follow the procedure to create New setup Vizard Protocol or Edit the existing 2 Protocols Protocol Manager channel Protocol Delete Copy select Cancel Protocol Summaries ioj x Current Protocol Select name tor new Protacal fast r channel with Z seried a Protocol Bright Fiel a Protocol Fura Ca 1 Protocol EYFP CYFF ff Protocol GFP DsRed H Protocol fast Insert Repeat Z action into the Tree Note you can insert in more than one location and get different effects As soon as you select the Repeat T from the Insert Item dialog and click OK you are switched to the Z definition Wizard to assign settings mZ Series options Set Start 48 55 A Stack Definition Bre Number of Planes C 2 Positions 2 Position xa LE 54 54 gt gt gt gt gt gt 207 Andor Technology Andor iQ USER GUIDE When you insert Repeat actions a matching End action will be inserted If you want to execute any actions within the Repeat loop you have to drag the End down the Tree You drop the action keyword End
77. Series Re Camera Binning Use Current Wait Allocation Off Camera Selection Use Current tie Events a AY Scan Test Compatibility Focus dP Pause Click OK to start protocol an Repeat T 60 times O mes fastest asi Auto Focus Activate Every 10 times SA Auto Focus D0 Repeat xY 4 Positions 3 g Repeat Channel Chl Che Ch3 HA Channels Description Repeat 10 umin 11 planes al Snap End Z End d End TO This is a regular Protocol for multi dimensional image acquisition By regular we mean that the frequency of acquisition is consistent for all fields Channels and Z values This kind of Protocol results in a single nDim because it is regular 215 Andor Technology Andor iQ USER GUIDE 4 6 11 Protocol Templates In order to make protocol creation easier we have added Protocol Templates These are special predefined protocols that allow the user to quickly create one of the patterns described previously When a new protocol is created a new wizard panel is displayed this prompts for protocol name but also allows a template to be selected Andor iQ 2 2 Andor Camera File Image List Settings Wiz Process Analysis Setup izard Protocols Several templates are provided Protocol Manager with the iQ installation Advanced users may create additional Please Enter Name for Protocol se 3 pa lene templates as described in the Appendix Protocol Templates Cur
78. This allows you to choose the Current camera edit the name of the camera and change the Image Window background color as a visual cue or reminder of the selected camera Andor iQ USER GUIDE 4 7 2 Dual camera simultaneous operation We assume here that the relevant hardware has been installed in the PC In this example we illustrate the use of two identical cameras iXon DUV 897BV E The screen shots shown here were taken from a system running the cameras with CCI 23 PCI and CCI 24 PCI e controller cards We want to perform FLZ operation for best performance so we need either an Andor low cost break out box BoB or Andor PCU for device control 4 7 2 1 Creating the Configurations Device Configuration Manager Open the Configuration Manager File Edit Settings Tools About Exit evel dla supplied with iQ 1 10 2 or later and New Edit Delete Exit create two configurations one FLZ whe which will be used for the so called 7 Created By FL2 master Creator Not Found Date Created 02 04 2010 1225 40 Master instance which generates the o Date Last Modified 02 04 2010 12 25 40 ap ea Acquistion Device Andor Camera With fast Piezo Z and Monochromator trigger signals from the PCU or BoB Xa TL slave and a second for the Slave instance 7 Created By TL slave Creator Not Found o Date Created 02 04 2010 12 27 05 in the Time Lapse mode as shown in o Date Last Modified 02 04 201
79. Time An example of where this Ratio two images or image series f Ratio two images or image series taking average of denominator method is appropriate iS outlined in Sell Ratio taking average of selected range for denominator Section 3 6 3 where a protocol is developed for baseline imaging Source Background Mask Calibration Numerator followed by compound UV uncaging Background i and then fast imaging of a single Mumerator U wavelength indicator The Threshold O Denominator Senne fluorescence fluctuations monitored in 325 this protocol can be normalized to the baseline level using the second F P 7 method The resultant series length will always be that of the numerator Andor Technology 383 Andor iQ USER GUIDE Method four not illustrated is applicable to single wavelength indicator experiments more than one indicator can be handled in one experiment where some period of the series is used for a baseline normalization similar to method three above The main difference here is that the image series contains both the baseline frames and the frames for normalization The user interface needs the baseline line frame range denominator to be specified Image Selection Image Details For the example image series Mame FURA TIF Type 12 bit greypseuda Intensity 1 000000 lus of the HEK cells we are 476 7 000000 pm rere interested in the change of
80. Tool can be found in Section 4 10 of the User Guide Acquisition Analysis AGE Analysis Calibration Movie Editor Spot Analysis Manage Analyse Review New E dit Delete Currentanalvsis Ploidy meme pot Analysis Plody A Setuplmade CAM DOCUMENTSIMAGES FOR TESTIN fee Bright Objects aa Lower Threshold 110 ak Upper Threshold 255 AD Area Filter 100 to 359 65 Andor Technology Andor iQ USER GUIDE 2 The Image Window The current image is displayed in the Image Window e Paula drosophila PBC_VerticalFlip_Rotate180 16 Bit 642 0 107 x 1020 0 107 4D File Edit Image List Region View Fast MIP Bw WA Alel DS Tl im L Time Wavelength 0 00 00 000 him s ms Frame Number Minrange Maxrange o Increment S Interval s ms a gt fo fo E f gt 00 100 Aid m i ao MEZEG Set Min Set Max w a Automap 1000 335 1 341 1444 iS e xx 500 i The Image Window will update the displayed image in response to several actions For example whenever the Live or Snap buttons are clicked on the Control Window or when the Run buiton is clicked with the Display Image option selected on the Acquisition tab The Image Window updates when you Show from the Image List or use the Open Image File menu When iQ opens from it displays the last image displayed in the previous session When on line Ratio processing is executed during a Protocol a tab named Ratio will
81. Triggered Events by name Picospritzer High Wash High Select Trigger Select Trigger Clicking the Add button shows the Trigger select dialog left from which you can choose the input s and states that will generate an Event As the Protocol Executes the Status box Acquisition Analysis RGB Analysis Calibration Movie Editor Spot Analysis indicates how many frames have been acquired from the sequence and among other information the number of Events will be displayed as shown here Edit Event Markers A XI NEE EET E When the Protocol has completed or Mi Picosprizention z Wavelength 1nmFieme S 185m stopped an Edit Event dialog is v2 JPicosprizerHigh Wavelength 1nm Freme 16 9957ms ghown which allows you to edit the text Ma PicospritzerHign x Wavelength 1 nm Freame 27 15517ms associated with the Events This is especially useful when you are using only the Spacebar for events and you need to differentiate Editing immediately after the measurement will make sure your records are accurate Enter names for the events 233 Andor Technology Andor iQ USER GUIDE Wavelength Time 0 00 04 261 hkmsms Frame Number Mintange Maxrange o Increment a interwal zma a 4 7 o 27 fi foo t ra m w w ft gt Set Min Set Mas Picospritzer High E Picospritzer Hic Go to Ewent O ae we Eicospntzer High f 2 Picospritzer High
82. View of an animation produced by FastMIP the OpenGL maximum intensity projection Viewer described in the next section Where movies are not suitable for presentation then Strip View provides another means and can be helpful in explaining the unfolding of events 305 Andor Technology Andor iQ USER GUIDE 5 11 The FastMIP and 4D View The FastMIP tool is accessed from the Image Window and has its own high level Menu item for direct access FastMIP uses OpenGL to provide maximum intensity projection MIP views of 3D 4D and 5D data during or after acquisition We use the term Fast because we rely on hardware performance for rapid rendering and also because we only load the head on XY view of the image data The result is that FastMIP can provide a rapid validation of experimental progress while having little impact on acquisition performance The Viewer is multi threaded to minimize impact on other processes An example of FastMIP at work is shown below with a 4D multi channel data set Note in 4D a Navigator allows playback through time to observe specimen development activity or signaling for example Fast MIP CLY 647 Maximum Intensity Sele Os Change Rotation Center Always On Top e Iv Multi Hn 00 00 04 405 himsims Frame Number Minrange Maxrange Increment Interval s ms r on Ae e e f focoo a aj ia mimi ie i ae Sethin Set Max FastMIP can work with data volumes that have Z or Time as t
83. Voltage to Normal The user can explore different configurations for their specific experimental needs Output Amplifier The Output Amplifier enables the construction of a more flexible camera The EM Port output can be used for extreme low light detection while the conventional output can be selected for maximum dynamic range operation If EM gain is used the EM port must be used and will be selected by default The Baseline Clamp option is on by default This setting ensures that the camera electronics adjusts automatically to deliver a baseline count zero light grey value of about 100 for iXon series 200 for Luca 500 for Clara and 1000 for original early IXon DV series 457 Andor Technology Andor iQ USER GUIDE A3 2 2 2 2 3 The Image Orientation Tab Flip done first The user can choose to modify the image orientation by selecting the radial button None Vertical or Horizontal This allows the user to view an image on the computer screen that coincides with the image in the microscope ocular independent of the ohysical orientation of the camera Rotation in Degrees done second Exposure General Temperature Shutter Speed Amplifier Image Orientation M Flip done firstly None C Vertical C Horizontal Rotation in Degrees done secondly None C Clockwise 90 C Anti Clockwise 90 co 180 This allows the user to correct the rotation of the image on the screen if the
84. a multidimensional image as a Image Import Options series of single files E g Select Common Name MyImage t0001 w0001 tif indicates that the image has both time and wavelength dimensions When the images do not meet this i Firstly you should select a directory that has all the image files you want requirement Image Importer to load in then type in a common name that is contained by the files Wizard In addition to some user fou can also specify a name for the destination image input can open them asa familiar Finally click the Next button to go to the next page multidimensional image for Common File Marne Destination Image Name Importedimage viewing manipulation and export Directory The first step is shown here D Kinetic COMMON T SSS m bantam dll 44 Andor Technology Andor iQ USER GUIDE 2 xj Pressing the H button brings up a Select a Folder Desktop a My Documents Sf My Computer H Local Disk C H a Local Disk D fl DVD CD RW Drive E E jwainwright on Oak users Z cy S My Network Places Image Import Options Set Import Dimensions Back Next gt gt Cancel In this page you need to check on those dimensions that the resultant image will contain IF vou have selected Mult Channels you algo need to choose the number of channels for the image Import Dimension W Multi Channel Number of Channels E FURA timen wavennini ti brows
85. an edge of the user defined region it displays the sizing arrows Holding down the left mouse button and moving the mouse will move the edge or corner in the direction of the arrow Right clicking on the image or a selected ROI will also bring up the same menus as discussed in Section 2 10 1 93 Andor Technology Andor iQ USER GUIDE 2 10 3 The Zoom Button Gr ala The Zoom button enables you to magnify the image displayed in the Image Window enabling you to view the fine detail of features of interest more clearly When the Zoom Icon is selected left clicking on the image will zoom in on the image centered about the point clicked whereas clicking on the image with the CTRL key held down will zoom out from the image centered about the point clicked The Zoom Tool can be used in conjunction with the vertical and horizontal image window scrollbars which enable you to zoom to higher magnifications and then scroll to the feature of interest The current magnification of the image is displayed in the text box below the Image Window expressed as a percentage value 50 represents a half size image one right mouse button click 100 represents a normal size image no magnification 200 represents a double sized image one left mouse button click Etc The Zoom tool zooms out from the image without the need for the CTRL key 2 10 4 The Auto Zoom Button ok ajaja When selected the image will be auto sc
86. axis BinningY binning of the Y axis Exposure exposure time in us All sections which name starts with Tab contain camera settings from the device setup window Image Info This section contains information relative to the camera s chip settings redondant with most of the fields from the Grab Parameters section 506 Andor Technology Andor iQ USER GUIDE Image Bits This section is for internal use only First Frame This section contains one field SavedTime containing the time stamp of the first frame Comments This section contains any comment added through the Image Manager To add user defined content select the Edit Comments menu item in the Image Manager dialog 507 Andor Technology Andor iQ USER GUIDE A4 1 2 Examples 1 The example is shown below for a 6 by 4 multi well image with 5 fields per well set in systematic sampling mode The wells 9 10 15 and 16 have been rejected Name TL 3Ch MF24 08 01 2010 10 06 01 AM Type 14 bit grey pseudo Intensity 1 000000 lux x 1004 0 196078 um y 1002 0 211172 um XY 5 Well 20 Created Date 08 01 2010 Time 9 57 08 AM Created End XYZScan ScanName Multi Well 6x4 5SysSampling FileVersion 2 Centre False Channel WellPattern 6x4 2mm 2 5mm plate Density 5 Calibration Manual XY Fields 100 8691 15017 124 83 9165 15017 124 83 8691 15724 124 83 9165 15724 124 83 9639 15724 124 83 11190 21405596
87. be added to the image list 405 Andor Technology Andor iQ USER GUIDE Math ND Image Once the processing is complete the new Destination Image will appear in the ImageList from where they can be shown and selected for other processing __ Display Current Image Slice BANANENICANANMANRONGAAN 406 Andor Technology Andor iQ USER GUIDE 6 13 Dual ImageDisk Plugin The Dual Image Disk plugin is found in the Process menu in iQ s Main Window It is primarily designed to combine images from two separate Image Disks For example a dual camera system incorporating two identical cameras running under 2 instances on of iQ on a single PC capturing data from two wavelengths or two imaging axes simultaneously Simultaneous capture can also be supported under two separate PCs each with its own ImageDisk The Dual Image Disk plugin can see the data on one or more Image Disks and combine them into a single dataset including alignment adjustments rotation and translation Dual Image Disk Local Disk Dynamic Image Disk CAIMAGES DYO V1 4S4FILTERSSOURE RUNT_250M5 TIF_Foallinge CAIMAGES DYO V1 4S4FILTERSSOURE RUNT_ 250M5 TIF_Foallinge CAIMAGES DVD V1 454FILTERSSOURE RUNT_ 250M35 TIF_Follinge CAIMAGES DVD V1 495FILTERSSOURE RUNT_ 250M5 TIF_FollingE CAIMAGES DVD V1 495FILTERSSOURE RUNT_ 250M5 TIF_FollirgE CAIMAGES DWO V1 495FILTERSSOURE RUNT_250M5 TIF CAIMAGES DYO V1 49 FILTERSS2D_GEL IPG CAIMAGES DYO V1 49 BRIGH
88. be ticked in the Options tab For each of the filtered planes specified during the Experimental setup e g for the red green and blue planes of a RGB image use the scrollbars to specify the minimum and maximum pixel intensity values that will be considered All pixels with intensities within the specified range will be labeled as containing that color component The specified limits are displayed in the 254 Andor Technology Andor iQ USER GUIDE graphs and the image in the Image Window is updated as described overleaf thus enabling interactive optimization 20 40 BO 100 120 140 160 180 200 220 240 Green Using the scrollbars select the minimum and maximum pixel intensity values to be considered for each plane Pixels are labeled depending upon whether their intensity lies within the specified range and therefore contains the appropriate component As you adjust the maximum and minimum pixel intensity values for the red green and blue planes the graph to the right of the scrollbars is updated and every pixel in the image displayed in the Image Window will be colored as described facing 255 Andor Technology Andor iQ USER GUIDE Red Green Blue Assigned Colour Yes No No Red No Yes No Green No No Yes Blue Yes Yes No Yellow Yes No Yes Magenta No Yes Yes Cyan Yes Yes Yes White Transparent the original image is No No No displayed For example all pixels with intensities that lie within the spe
89. being used To save the current settings to file press the Save button This will open a standard Windows save file dialogue from which you can specify the filename drive and directory in which to save the current settings The settings can be reloaded at a future date by pressing the Load button The Zero All Axes command is used to reset the stage as described previously in Section A3 5 1 above 473 Andor Technology Andor iQ USER GUIDE A3 6 Ludl XYZ Stages and Filter Wheel Andor Technology provides support for Ludi XYZ stages and filter wheel and also for ASI XYZ Stages Section A3 10 These devices have similar configuration and setup so they have a common setup description for the Device Setup and XYZ stages A3 6 1 Device Setup The Ludl and ASI device setups are accessed from one of the Device Device Setup Setup buttons located on each window of iQ The device profile must first be setup in the Configuration Manager When the profile is selected the device setup will have a tabs labeled accordingly In addition to the tabs the following Options are available at the top and bottom of each Tab Current Channel is located at the top of each tab under Device Set up This allows the user to go live or snap and image with a channel by selecting from the dropdown menu and the record the changes to the channel settings Acquisition Auxiliary Devices Record 474 Andor Technology Andor iQ USER GUIDE
90. below 288 Andor Technology Andor iQ USER GUIDE File Plots Data View Cell No Frame Area Perimeter Max Chord Shape Int Int Mean Int Log Int Int_ a Cell59 15 894 000 134 000 39 000 0 626 66863 000 74 791 11 110 Cell60 15 757 000 130 000 42 000 0 563 63747 000 84 210 11 063 Cell61 15 633 000 118 000 34 000 0 571 36182 000 57 160 10 496 O Cel62 15 1261 000 166 000 50 000 0 575 116156 000 92114 11 663 Cell63 15 23 000 110 000 34 000 0 543 36507 000 69 803 10 505 Cell64 15 758 000 130 000 39 000 0 564 66491 000 87 719 11 105 Cell65 15 749 000 134 000 44 000 0 524 57678 000 77 007 10 963 O Cell66 15 1384 000 198 000 63 000 0 444 133665 000 96 579 11 803 Cell6 15 1166 000 158 000 45 000 0 587 119869 000 102 804 11 694 Cell68 16 903 000 148 000 43 000 0 518 76315 000 84 513 11 243 Cell69 16 698 000 120 000 34 000 0 609 63339 000 90 744 11 056 Cell 70 16 1052 000 152 000 50 000 0 572 103338 000 98 230 11 546 Cell71 16 779 000 130 000 37 000 0 579 36473 000 46 820 10 504 Cell72 16 823 000 130 000 36 000 0 612 67205 000 81 659 11 116 Cell 73 16 760 000 130 000 39 000 0 565 64076 000 84 311 11 068 O Cell74 16 3504 000 300 000 84 000 0 489 19853 000 148 360 13 161 x Cell 7A 1A 848 000 14 NNN 44 NNN NAA 7A9 n NNN AD AR 11 238 NS Total Spots 102 Histogram Scatterplot Save Data Clear Selection X Cancel Included Spots 99 All Data C Single Frame The check boxes to the left of each row can be used to mark a
91. cancel an Experiment before completion press Stop The Channel button opens the Channel Manager Section 4 2 which allows the setup of channels Channels describe how your hardware devices are set up to illuminate and expose the sample Click on XY Scan to open the Scan Manager Section 4 3 which allows the setup of scans These are used to analyze samples at more than one field of view in the XY plane and or at different Z coordinates in a Z series A scan pattern defines at which XYZ coordinates the sample will be illuminated and exposed The Device Setup button opens a dialogue in which all the hardware devices 59 Andor Technology Andor iQ USER GUIDE included with your system can be set up and configured Section 2 3 8 Loop Protocol is toggled on and off by clicking in the checkbox When selected the checkbox is labeled with a tick and on completing the Protocol it will repeat using the same specified criteria overwriting the previous acquisition To end the Protocol press Stop lf Loop Protocol is deselected the current Protocol will run to completion and then the system will return to Idle A possible application for Loop Protocol is where you want to capture an event whose starting time is unknown Simply waiting for the event to begin and then pressing Run would result in you missing valuable data at the start of the event You can therefore leave the Protocol Looping ensuring that you capture the whole of
92. cells marked for deletion Cancel returns you to the Review panel dialogue 294 Andor Technology Andor iQ USER GUIDE These options can also be accessed from the menu bar as illustrated below gem Plots Data OF Cancel Clicking on the Clear Selection button ignores the selected cells removing all the red crosses from the image gallery File Plats Clear Selection Print Spots Clicking the Print Spots button allows the image gallery to be printed to enable a visual comparison of all detected features Image Print Setup Select Layout for Image The print layout can be adjusted to specify the Total Number of Images 15 number of columns and rows in the printed Number of Columnsper E page A Humber of Rows per page 5 i Number of Pages 1 Cancel 295 Andor Technology Andor iQ USER GUIDE An example Print Preview window is shown below Kinetic Print Preview Iof Printer WWEB SERVER Brother HL 1 Setup Pita Orientation i Portrait i Landscape j RR RAR RR RAR RARA ARA RARA RRA RAR AA RA AR UU A RARA UU A BAR RU nnn i ls ea 5 inet p Prey aad Preeerer rer eerrrer rr rrr errr rrr errr ire rrt errr rrr er rere Beset Magme M View Margins 5 7 6 3 Histograms To plot a histogram click on the Histogram button or select the histogram plot function using the menu bar as shown previously A parameter selection panel is displayed as
93. create button will show processing activity 5 8 2 Kymograph Results Window Once completed the right side of the Kymography Window will display the results in three different formats Kymograph Line Profile and Plotted Data Click on the corresponding tab to view the results 5 8 2 1 The Kymograph Tab The Kymograph Tab shows the changes in relation to the line selected Press the Snap button to snap a new image Kymograph Line Profle Data Press the Tracking button to track the plots through the series CORKS ees Tracking The user can look at multiple channels at one time as shown in this example or look at single channels To look at a single channel simply select one of the colored boxes relating to the palate chosen for that particular channel M Multi B KI Andor Technology 313 Andor iQ USER GUIDE 5 8 2 2 The Line Profile Tab Kymograph Line Profile Data The Line Profile Tab shows the plotted line through the Kymograph In this case both channels are plotted Pline chi 500 ee E aPline cho Selecting the Edit button will open two options Only the profile of the fist image in the sequence is shown Scaling Highlighting Scaling will give the user the options of Auto Manual or Full Range Select the appropriate option to apply to image melee w AUCO Colours Manual Full Range Colours Highlighting Colours will give
94. device associated with each of these You can use the same device for both Reference Z and Scan Z Camera Alignment This allows you to co align two cameras and in combination with the Field Split plug in allows for co alignment of data from two cameras This Wizard is described in section 3 4 along with the Field Split for dual camera systems More details on Field Split for single camera use in provided in Chapter 5 Stage Alignment Camera and motorized stage orientation and motion alignment necessary for correct montage and multi well or micro plate scanning Follow the details in section 3 5 Select FRAPPA Device Choose which Channels are FRAPPA channels to help iQ identify their special needs Note in general iQ 1 X only controls the laser combiner and FRAPPA scanner in these Channels FRAPPA Calibration FRAPPA calibration helps you create a correspondence between the camera pixel positions and the FRAPPA scanner angular positions This allows you to bleach and activate user Andor Technology Andor iQ USER GUIDE 104 specified ROIs See section 3 6 for a description of FRAPPA and FRAPPA calibration Channels the Channel wizard helps you set up and record the state of all the instruments in the system to acquire images for specific modalities or fluorophores e g DIC epi EGFP CSU mCherry TIRF CFP More details can be found in section 3 6 Please note that the exact sequence and form of the
95. eai a aaa raeas 320 S10 The Strip View Toolen aee aa Aaaa aa aea a eaaa 323 S V The FastMIP and 4D Vie W oerni Ea 326 Dol ZIVIOITAGE VICW aaeoa Oe EOE A S ES NO SEESE 329 SID ERAP ADAl y SS deo A ATA r AAE Er A EAER 334 6 PROCESSING TOOLS iira a Eaa Eaa cb a A EO UE a Ea a a ala nao e EO N te 340 6 1 Process Episodic A Vera DO asc scoscsssiecsscscousnccscaesssavescavessuscsuctpeseayesseeusussseassusasscasustpeswsussneaeseeassexstossstesesepesuaes 341 02 Mask Wizard sd E a A A ALUDE DOU A DU UI Dn E A ALU DE EUA a E E ALU DEU LEO EDU Una 344 0 3 FTC LOS Pla ATA AA scnetenencanin E sh scmatonaigas cseessuasoasceass aussie 345 GAT ROUGE ATAN ATA a DNA NU O EOE e EE TEO 355 63 Image FEE yine a E E E E A TERE RE 356 6 6 Rolling Ball Fiter ATA AA AA EEE 360 3 Andor Technology Andor iQ USER GUIDE 67 Threshold Tma O sco dec cies i Somcas Cacccu AT a aS 363 6 5 Clear View DECONVOIITION givscsiostcsecssnsceussictsouss a a tescecensespoussbensewesencsceopsepeaeass 364 09 BOSE Essa EE aa E RES A ERE RS AS RESE ASAS ERAS 366 6 10 The Image Math PrOCOSS Vania TA EEEE S AE TADE CAETANO 370 OTERAO MA CO AA AA 378 6 2 Image mae Math PIM OM escecceseistolsccceexsesgessoescecseevesadstocaccasexeesecetaussvuseussiqds ver ssersouceleceseeadeseaaensecseaveceretes 396 615 Dual imase Disk PIMGI jicccdsccesesesvcgoicececavevevceccsseseassscescaasesesseaseceveuseaseseesseceswossecesscoveseseusaboceeeseesessegeececsous 407 6 14 Pl sg In ReGistv ation and ACCESS
96. easy to use solution to a class of image analysis problems commonly found in bio and industrial imaging We hope it proves useful in your studies Analysis may be performed on images acquired using iQ or previously captured using third party software and stored on disk 5 7 1 Analysis of Images Acquired Using iQ Spot will analyze images captured using iQ in the following ways Analysis of a single image captured using the Snap command Please refer to Section 3 4 2 of the iQ user guide for further details A batch set or group of images for cumulative analysis such as may be obtained from multiple fields from the same sample to ensure suitable sample size or spatial sampling Please refer to Section 3 3 of the iQ user guide for a complete description of the multiple field sampling functions A time series of individual image files or a multi dimensional time series image for analysis of spot feature dynamics Please refer to Section 3 4 4 of the iQ user guide for a description of how to perform a time lapse experiment To obtain accurate analysis data a valid XY calibration must be used during image capture and further details concerning the calibration of images can be found in Section 2 7 4 and of the iQ user guide 275 Andor Technology Andor iQ USER GUIDE 5 7 2 Analysis of Images Acquired Using 3rd Party Software The following image types can be imported into the iQ for subsequent Spot Analysis An individual ima
97. execute in FLZ mode New Protocol SiGe RSS When you click OK the prototype FLZ Protocol Slverast shown on the next page will be created v Fast Time Select Fast option if a you wish to stream single channel data at maximum speed 225 Andor Technology Andor iQ USER GUIDE Acquisition Analysis RGB Analysis Calibration Movie Editor Spot Analysis You can now edit it the Protocol Status o x tos Live to change the Channel from oa Snap Current number of Repeats Save and Interval Pause message etc Curent as described elsewhere in the Auto Save j i C Andor i Data Ishihara Lab FL2 master Autolmages eS manual Acquistion Calibration a ES Optovar Select 10 None Selected a Protec i Protocol Gd Image FLZA Protocol fF Protocol Type Fast Time Camera Binning Use Current Q Wait Allocation On trun a Camera Selection Use Current Channel Pause Click OK to start protocol OS Interval O ms fastest Scan Repeat 1 times Channel Current Device Setup n Snap End Loop Protocol IV Display Image Save As Options Ui 4 7 2 5 Setting Up the Time Lapse The Time Lapse Protocol needs to be a simple Fast Time Lapse setup with a single channel Note that the cameras settings for readout and exposure need to match or be faster than those of the Master otherwise the Slave will
98. experiment was carried out with an initial loop of 10 snaps in order to establish a pre bleach It then obtained 50 Z stacks in the post bleach A FRAP region and two reference regions were defined on the image overlay The tool also allows the option of overriding any historical region data on images loaded from the image disk Remove any historical regions and create new ones in the desired locations The dialogue for this experiment appears as below in Protocol Protocol Florian Muller National ey Image Protocol a Camera Binning Use Current T Wait Allocation Of Camera Selection Use Curent Hong Events an Repeat T 100 times O mg fastest al Snap Frame d End T ir Frappa frap Mosaic Selected FRAPPA gi Repeat T 1100 times O ms fastest 2s Move Scan Z 26 Repeat 38 um in 2U planes Snap Frame dW End 2 d End T Institute of Health Fluorescence Imaging Group Andor Technology 335 Andor iQ USER GUIDE 2 FRAP Analysis C DOCUMENTS AND SET TINGS P BINGHAMWDESKTOP FRAP_ANDORTIFF TIF Mobility Chart xX Axis Mode Pre Bleach Correction Point Position Zz FAI 4 E gt 26 Region Selection x FRAPPA Reference _FRAPPA_1 FRAPPA 1 _FRAPPA_1 _Reference_2 800 900 1 000 1100 yO 4 Point Data Average Values Mobile Fraction 0 643 Half Life ms 43 631 Diffusion um 2 rms 0 886 Frappa Region Reference Region Beam Width um Calculate _FRAPPS_
99. field during field selection for proper operation e Autofocus support currently includes Olympus ZDC Nikon PFS and Zeiss DF systems When you decide to create a new scan pattern enter its name into the text box Make sure you use a descriptive name to aid your memory if you wish to re use them in future Select the appropriate Scan Options by enabling the relevant check boxes Press Next to continue As examples using the Scan Wizard we illustrate creation of Multi field Multi well with well rejection list and Montage scans We hope that with a little practice the Wizards will become easy to use Note on Z Scans iQ2 handles Z scans or Repeat Z Actions differently from previous versions Although only one XY scan can be used in a Protocol Tree it is possible to use multiple Z scans Whenever you Insert or Edit a Repeat Z in the Tree you will be taken to the Z scan Wizard to define Start End and Step size of the scan as shown in section 4 3 2 When you click Next you return to the Tree view Note that ALL Z scans are relative not absolute The currently Z position is used as the center of the Z scan rang 162 Andor Technology Andor iQ USER GUIDE 4 3 1 Flow Chart of Scan Setup and Options 163 Andor Technology Andor iQ USER GUIDE 4 3 2 Z Scans P Andor iQ 2 2 Andor Camera x If you Insert or Edit a Repeat Z Action File Image List Settings Wizards Process Analysis Plug Ins Help Setup wizard
100. from the Depth Dim combo box Time as indicated by the arrow in the figure above on the right 5 It may be easier to see all changes that happened to the cell or membrane during acquisition time by displaying the source image in Maximum Intensity Projection fluorescence or Minimum Intensity Projection ophase DIC mode Try using these modes with your data Note some LUTs will provide better contrast than others 6 To start the analysis process select the Point Cross button from the tool bar Place a cross to mark the centre of the cell you are interested in 7 From the tool bar press the Line button and draw several lines from the centre of the cell to the outside region of the cell radially as shown in the figure below The direction of drawing is important as the Kymograph will reflect the start to send direction of the line when created Try to make the line perpendicular to the features you wish to analyse 316 Andor Technology Andor iQ USER GUIDE exea N 8 Clicking on the Create button will generate a Kymograph Lamella image and display it in the right pane as shown in the figure below 9 If you have drawn more than one line there will be a scroll bar in the controls region at the top of the RH panel Scrolling will show the kymographs corresponding to the lines drawn on the cell in the source image 10 To start analysing the protrusion contraction rates select th
101. hardware products Chapter 2 delivers an outline of the way iQ can be configured for multi user scenarios and the roles of administrators and users via the Configuration Manager It also provides a condensed review of the iQ user interface windows dialogs and controls Chapter 3 focuses on Wizards which are used for Calibration Alignment and Setup of instrumentation prior to acquisition of quantitative image data Chapter 4 addresses acquisition explaining how Channels Scans and Protocols are 14 Andor Technology Andor iQ USER GUIDE created and ends with a number of examples from ratio imaging to 4D with FRAPPA actions Chapter 5 provides details of how the Analysis and Visualization tools operate and what you can do with them For more heavy weight analysis and visualization we recommend Imaris Chapter 6 looks at the Processing and Analysis tools in iQ Appendices 1 and 2 address Installation and Configuration Manager in more detail Appendix 3 includes detailed explanation of some of the hardware device interfaces and their operation in Andor iQ Appendix 4 explains how metadata is stored in iQs multi dimensional image inside How To Documents are just that recipes on how to achieve a certain goal with Andor iQ 15 Andor Technology Andor iQ USER GUIDE 1 5 Andor ImageDisk Technology ImageDisk is Andor s image oriented virtual memory system which is optimized for the capture and treatment of lar
102. if the bleach is still not visible This step of finding the initial bleach may be tricky the first time you calibrate the system Once the bleach spot has been identified in the field of view then you must create corresponding pixel coordinates and angles for the calibration procedure To do this for each bleach point region click on your best estimate of the region center with the marker and then click on the button labeled Add Calibration Point The Mosaic device requires a minimum of 3 points for an adequate calibration When you are satisfied that you have sufficient points over the image then click on Next and the FRAPPA Calibration will be created by the system and used henceforth whenever a FRAPPA action is requested demanded 5 Setting the FRAPPA focus control Now that the FRAPPA system is calibrated in X and Y you can use it in FRAPPA 145 Andor Technology Andor iQ USER GUIDE on Demand mode This means you can go live and use a FRAPPA region type with a FRAPPA on Mouse Up action Place a FRAPPA region on the focused specimen image and right mouse on the region Select the FRAPPA on Mouse Up option when selected it will be checked Assuming the calibration above is accurate when you mouse up after moving the region the area immediately below the region in the specimen will be bleached Finally perform a series of FRAPPA tests within iQ making sure to test points a
103. immediately after the selected event Cancel Select Trigger a Select Trigger On selecting Trigger In the box shown left will appear Select the trigger input line in this case input O In0 Select whether the event will be triggered by the line in a high or low state NN Cancel The set up is identical for an output trigger You may wish to put more meaningful descriptive text labels associated with the Trigger lines This can be done through the Trigger setup panel Note that restricted users will not be able to change the settings but other users will 230 Andor Technology Andor iQ USER GUIDE Acquisitions Auxillary Devices X The Trigger setup panel is available through the Device Setup dialog Input Cutout Set Up Co ieee ee ee ee ee accessed by clicking the Device Trigger Hame Jaxon Trigger Setup button To make changes to States the panels you must click the Tfh on o Lock Unlock button at the bottom right BA Or o of the panel High Event re Low Event rey Then the Input Output lines are selected from the tabs Here a Trigger IN Input 0 has been selected to start acquisition in response to a signal from the Axon trigger device To make this clear the Trigger name has been set to Axon Trigger Any or all of the IO lines can be used 0 7 and labeled in this way The state or transition of the input line can be set here also The States High and Low have be
104. in the series is processed Ratio tool computes the maximum intensity in the ROI and subtracts it from all pixel values in the frame to remove the background This is done for the 340nm and the 380nm images independently so each is background corrected from the image of the appropriate wavelength 6 11 4 Mask Clicking on the Mask tab reveals the following panel laixi The Mask operation is used to reject File About SA VES ED ratio data or image pixels that lie in Mask Options Masked pinels set to value m background or low intensity regions of Mask where after background subtraction the image where noise and flare and Numerator lt o Denominator lt O other artifacts can give rise to spurious Use separate image pissin results The user can define to what value the rejected or masked pixels are Create Mask Image set by entering the value into the text box provided at the top of the dialog There are two methods available to apply masking The first is to ignore Source Background Calibration Mumerator Background Region Mas 1 Denominator Background Region Max 1 pixels with a low value e g 0 as shown here but you may wish to set it to some Numerator Threshold 0 Denominator Threshold 0 value well above the noise expected after background correction Depending on the image detector used this may be from 5 50 grey levels Ok Cancel Help
105. into the Protocol Tree you will be Setup XYZ options directed to the Z Scan Wizard to set up the Z options Select Z Device Device Setup Next gt gt re Series options reading the correct Z device via the First check that you are controlling and Setstan 1055 Select Z Device button There may be sacana fass a Piezo and a microscope or stage o control Z motor Choose the one you AZ ur PESO Nyquist Cubic want to use in the Scan Z selection The To a position reading of the selected device pStack Definition will be displayed live in the dialog Z C AZ Number of Planes C 2 Positions positions m2 Position EEEIEE 54 54 x 33 555 When you enter the Wizard dialog iQ switches into Live mode You can now focus up and down manually to discover the upper and lower extremes of the specimen Z range of interest If you want to switch to a different Channel or access adjust the Z value through the Device Setup dialog then you can click on the Device Setup button Manually adjust the Z Motor or Piezo to the starting position of the Z Series then press Select Start The Z coordinate at this point will be recorded in the text box There are three further parameters on this dialogue You must enter the values of two parameters and the value of the third parameter is then calculated Select the parameter to be
106. ipp20 3 Microsoft VC80 CRT Microsoft C80 MFC O Templates O Common FilesiQ 2 2 Testing D Does 7 90 KB 4 My Computer a ype KI_EXP File Date Modified 12 13 2010 9 36 AM Size 7 90 KB Andor Technology Andor iQ How To Documents Now when you attempt to create a protocol a new item will be present in the template list Andor iQ 2 2 Andor Camera 5 xj File Image List Settings Wizards Process Analysis Plug Ins Help Setup Wizard Protocols Protocol Manager Please Enter Name for Protocol select Cancel Protocol Templates Current Protocol MyTemplate 4 Protocol Time Lapse co i Protocol MC H H Protocol MCZ Z First 1 4 Protocol MCZ 1 9 Protocol MultiPhase 4 Protocol MultiPhaseWithFrappa 4 Protocol MultiRate E Ai Protocol My Template Py Protocol Ratio 571 Andor Technology Andor iQ How To Documents A5 21 Configure iQ with Lumencor Light Engine The Lumencor Light Engine LLE is a high performance solid state multi line light source which uses LEDs to pump luminescence in doped crystals It can be considered as a super luminous source The emitted radiation covers up to 7 spectral bands in the near UV Vis NIR regions of the electromagnetic spectrum as shown in the figure below By selection of integrated filters the emission spectra can be tailored to fluorophores used with fixed and live cell specimens NI A 450 500 550 600
107. is opened Calibration by pressing the Calibration Manager button on the Calibration XY tab or selecting Calibration from the Wizards Edit Delete Close menu Calibration Summaries Here we show the Calibration Manager Current Calibration 887 iXon 10Xwi XY Calibration 887 Xon_10Xwi cameras and a range of objectives E E xY Calibration 887 iXon 20X ag XY Calibration 887 iXon 40Xwi a XY Calibration 887 iXon_B0Xoi l l H 4 XY Calibration 887_10Xwi From this dialog you can create New H XY Calibration 887 20X E E xY Calibration 887_40xwi H LP sel nni BBO Button Edit and Delete calibrations Pe ee Click the New button and you will see 2 H XY Calibration Luca_40x i a a XY Calibration Luca_40Xwi options pop up choose Automatic it Calibration Manager with an existing set of calibrations for two a XY Calibration Luca_60Xoi you are sure of your scope and C mount magnification Choose Interactive if you are unsure Either way the Wizard will guide you Here we illustrate the Automatic method Please note that the Default calibration present when the system is first run cannot be deleted It is replaced on the creation of a new calibration file 106 Andor Technology Andor iQ USER GUIDE Nee aa You should create a new Calibration set when you first set up your system adda Objective Selection
108. lel w omarion G Display Every Fiame C Display Frames s Actual Frame Rate 11 52 Experiment Control FLZ trigget PCU DO 7 F Falada Avail Memory MB 2047 Trigger Control coe Tet TT me Festin I existan st Tage Aval Hen 2047 OK Display Corbol Display Every Frama Display 5 Frames s Actual Frame Rate 11 58 Timeout 1000 ms C Fs hun 7 Be Ste Eat Tr Camata Status Camera ts working 4 7 2 3 Setting Up FLZ Devices and Sequence To setup up the two instances of iQ you can run the Master in either Time Lapse or FLZ mode but the Slave must be run in Time Lapse camera only mode because we do not want it to try to control hardware the hardware like the Master Before you set the Master into FLZ mode to make sure that the FLZ devices are connected You can now proceed as explained in the FLZ section to configure the hardware and setup the Sequence tab for the desired acquisition sequence When you have the above devices selected you will have controls enabled in the Sequence tab as shown below and the properties will reflect the device settings from the Wavelength and Z tabs 224 Andor Technology Andor iQ USER GUIDE Acquisition Auxiliary Devices omen _tastve tyson A v nv k Snap B ecos new e Note that in this case we have selected Camera Selection Camera simultaneous excitation with wavelengths DUS97_BY 3877 Settings Experiment Seque
109. mathematical processing is started Background correction will subtract the value specified in the selection from each pixel in each image prior to the calculation Options included are A constant value can be specified Maximum or Mean of a numbered Region of interest One frame from the Source Image s A B A separate image in the image list can be used Select one of the options and type a value into the text box for both A and B if applicable If Use separate image is selected use the select button to choose an image from the image list 6 12 3 The Mask Tab Source Background Mask Mask Options Masked pixels set to value ct o f Mo Mask CO Mask where after background subtraction A so B oo C Use separate image Mask Image san Create Mask Image 403 Andor Technology Andor iQ USER GUIDE A Mask defines the region over which the processing will be executed It is a binary result of a pixel by pixel comparison against a constant value Mask where or it is a binary image or image series Whenever the result or image is 1 the calculation will be executed otherwise the pixel in the Destination image will be set to Zero This ensures that processing will be constrained to regions of the image where there is useful information and not background noise Select No Mask if a mask is not being used Select Mask where after background subtraction if you know the values you want to use Selec
110. miss trigger signals from the Master and drop frames The other thing you must make sure of is that the number of frames to be captured by the Slave is equal to the number captured by the Master You can compute the number of Master FLZ frames Nm as follows Nm Np Nb Ch Zs Where Np is the number of Repeats in the Protocol Nb is the number of Bursts in the FLZ Burst setting Ch is the number of Channels in the FLZ L setting and Zs is 226 Andor Technology Andor iQ USER GUIDE the number of Z steps in the FLZ scan 4 7 2 6 Notes and Cases where synchronization may fail 1 The Slave must be set to Ext Trigger mode and it should be triggered from either the Master Fire pulse or from the PCU or BoB DO7 signal If you fail to make the D07 connection iQ will eventually Timeout while waiting for a trigger signal and show the following dialog No trigger Time out in Ext Trig mode Please check cable connection 2 There are some circumstances where FLZ resynchronizes and dumps frames especially in Frame Transfer mode and with Bright field capture Burst settings where the Slave will lose synchronization because it has no knowledge that frames are not valid Thus the Dual camera mode is aimed at fast acquisition where the two cameras are operating with a wavelength or polarization splitter or from two different modalities Provided that the FLZ mode is not in FT mode or using Bright field then the cameras
111. mode is not suitable for use with the Andor FRAPPA device This device relies on the Active Blanking capability of the ALC to draw shapes but the fire pulse will be high when the FRAPPA device is firing Also Stream mode is not suitable when using the ALC with large delays between Snaps e g use of intervals The sample may be over bleached by the continuation of the fire pulse when iQ is paused in the interval 200 Andor Technology Andor iQ USER GUIDE Snap Frame mode is recommended to ensure correct behavior for these scenarios Note The camera Fire signal may not reflect exposure state when running in Frame Transfer mode If this is the case then include Trigger and Event device control in the Configuration and ensure that PCU or BOB DO2 is set to High or 1 at Preframe and Low or 0 in Postframe Couple the DO2 signal to one of the Active Blanking Inputs on the rear of the PCU for Laser combiner or other fast ms shutter input on other sources Return to Table Trigger In Wait for a Hi or Lo TTL level on a user selected bit from 8 TTL inputs The digital pattern is shown in the Tree as two groups of four binary patterns XXXX XXXX corresponding to the TTL inputs DI7 4 DI3 0 The hardware TTL inputs are handled by either the Andor PCU100 series or the Andor BOB 100D or BOB 100A interface modules You can use these inputs to synchronize iQ execution to external Events and monitor external hardware Return to Tabl
112. name for the new Channel and Press Next to continue Andor iQ 4 Sel e ct th e File Image List Settings QC Analysis Process Help E File Image List Settings QC e Setup Wizard Setup Wizard appropriate Setup Camera Setup FW and Shutters Filter Wheel and Auto Bue for Auto Blue ohutter se Nono lt lt Back Next gt gt Cancel oyu Ludl Shutter 1 ODpen Closed Ludl Shutter 3 p O S It O n S a n d Ludi Shutter 2 Current Ludi Shutter 4 fc Z Wheel Settings LIVE SelectLUT Grey X press Next to r Exposure Control continue Binning EM Gain Control G z O DAPI o 1x1 s dag fo a 440 nm 2x2 C 8x8 A Exposure Time l Shutter Open Auto Exposure Upper Limit Pue o 100 50 ms 0 Be e 10 C ws se M xO Y 0 Z 444 Actual it WHERE XYZ Readout Time ms AN 2 N 2 8880 Command Status z Initialization failed _Reset 5 Set up the Camera settings by choosing the Look up Table LUT associated with the Channel 6 Select the Binning and Auto Exposure then adjust the Gain Control until the desired image quality has been reached Press Next to continue 530 Andor Technology Andor iQ How To Documents File Image List Settings QC rd Help Setup Wizard Channels Channel Manager O _New Edt Copy Delete _ Close The new Channel will be listed in the Channel Channel Summaries i A i arianne Summaries and availabl
113. on the navigator window Change the Z dimension until the best focused slice is found and press the Set button on the Dialogue box Do this individually for each channel in the series as shown in the figure When all channels are done press the OK button to finish this dialog as shown Now you will be presented with the same Dialogue as in the case of single channels do it step by step as before for all time points and finally press the Create button and that will generate a new image for you which has only one point in the z dimension 369 Best Z gt Channel Offsets 7 DEVELOPME ES As tis de a multi channel image the best z slice might be different for different channels Pou need to select the best focused z slice for each channel in this page first and the offset info will be used to generate the final output image later m i E 1 ch Z set as 10 Clear All Cancel Andor Technology Andor iQ USER GUIDE 6 10 The Image Math Process The Image Math Process is accessed from the Process Episodic Average Menu Image Math operations include addition Mask Wizard a a Field Split subtraction multiplication and division between a source sip motste Image Image Filter Rolling Ball Filter image These operations also include Maximum and Threshold Image Clearview Deconvolution Minimum intensity projections MIP s which are useful in Best Z Image Math Ratio Image image an
114. on top of the action you want to appear after the End The example protocols below show inserting a Z scan and dragging the End to encompass several tree elements On the left the Z has been inserted above the repeat channel It does not contain any acquisition elements On the right the Protocol Tree is shown with the End of the scan being dropped into position End Z is located after End Channel after completing the drop Py Protocol Mult phase MEAT ma Image Multi phase MEAT a Protocol Type Time Series a Camera Binning Use Current Wait Allocation OF f Camera Selection Use Current Events Qe Pause Click OF to start protocol Ga Repeat T 100 times 10 sec Repeat 172 7 umin 12 planes End Z Repeat Channel CAT Che H Channels Description al Snap di End Channel OnLinelhart Pf Protocol Mult phase MEZT sa Image Mult phase MLAT a Protocol Type Time Series ait Camera Binning Use Current Wait Allocation Off Camera Selection Use Current Events dP Pause Click OK to start protocol ap Repeat T 100 times 10 sec Repeat 12 17 um in 12 planes Repeat Channel Ch1 Che H Channels Description al Snap Note the Z scan uses relative Z values 12 1 um in 12 planes and by default iQ2 assumes the current Z position before and after the Repeat Z is the middle of the scan This then allows for more than one Z scan with different r
115. orientation Microscope optics and slide debris apparent Select the Analysis menu Line Profile to show the ae egion eee r a edge transition in the o X1 1 LAC fo faa vertically separated but Line Width a horizontally similar lines ez 5 T Automa ROI O Jw a make sure the Live mode is used so the line profile updates with the Live image The graph on the next page shows the typical profiles for the two horizontal lines If the orientation between camera and DualView is perfect then the 2 pairs of lines should show almost identical black grey transitions Line Profile Focus Ioj x Fie view Edit Mode 1900 1800 1700 1600 1500 1400 1300 1200 1100 r yellow The above picture shows the Line Profiles color matched to the lines for ensuring correct orientation of the Camera and Field Splitter If the aperture of the Field Splitter is not parallel quite common then there will be a Andor Technology Andor iQ USER GUIDE small shift between the edges in the two profiles and the width of the light area will be different at the different vertical positions as shown above for a real device Now you need to ensure that the transitions occur as stated in the DualView alignment procedure at 1 4 and 3 4 positions of the chip width In the case of a 512x512 sensor as used in the iXon 887BV then these should coincide with pixel number 127 and 383 You
116. osecescccccservcesssececocevesesvawnccccovnssseseewsacecsensacecosesceccousetecs seus dceuseueeveasecetouees 409 GAS RGB Mero t sidos aiara E A AE ADE E IE E O dasessasseveiscsdocedeatenvesasedesessacs 410 6 16 Photo pleach COMPENSALI N rcesssccsscisctestecuensasisceeetis vevcavesicetentecasesxelercwabeatestevsasecteativecdasehesceedeatestouaniarneneer 413 APPENDIX 1 INSTALLING THE SOFTWARE oanunuuaaa anana a anana aun Ra a ana RR a URDA a DURAR DURAR DURAR RUDA RR a DU bodes A DD Das 417 AF Installation OF Sentinel Driver Ssa a Ea Aa 418 A1 2 Troubleshooting Dongle Issues sssssssecccccssssececccssssseecoccsseseeocccsssseccescsssseceecosssssceecsssssseecossssssseeeossssssee 421 A1 3 Initialising a Dongle for Additional Products sssseecccsssssececccsssscecoccsssscccocosssssceecossssseecocsssssscecsssssssee 424 A1 4 Install the Latest Windows NT Service Pack ccccsssscsssssscscccccssssccccccssssccccccscsssccsssscccccccsssccsscees 425 A1 5 Installing the Windows Scripting Host cccccssssssccccccccccsssssssssssssssscscccccscssssccceesssssssssssccccscssssees 426 A1 6 Installing your Andor SoftWare sisscciscccecsscssssscisedesossrosndecesessccdassovesedsecosesesesssnessesascdeussnsuebsesesesenoessssacesusee 427 APPENDIX 2 CONFIGURATION MANAGER ccccccccsssseeeeeeeesseeeeeeeenseeeeseeenseeeeeooenseeeseooenseees 428 A2 L The COonmoeuration Wana ger panca TA TA N 429 APPENDIX 3 HARDWARE DEV
117. ou have Administrative rights to this software Please selectthe Configuration for this session meteortrigger meteor sutter meteortriggar meteorte st meteortrigger No Grab No Grab SY LE SWAY Lie v Kinetic Trigger Device Kinetic Trigger Device E z Cancel Cancel After pressing the OK button you will be asked to specify a default path for saving analysis data Select Data Path select Default Path for Saving Data CAKinetic iQDatal Browse MB This can be changed at anytime using the File Set save Path menu item Do not show again 23 Andor Technology Andor iQ USER GUIDE 2 2 2 The iQ 2 User Interface Image Window p paula_drosophila tif__RunningBrightest 16 Bit 642 0 107 x 1020 0 107 4D Menus File Edt Imagelist Regon Wew Fast MIP ards Process Analysis q Hala x aaja Alel goos T ima a EA 7 a E iQ Control J s Menus en av m age To O lb ar Acquisition Analysis Movie Editor Spot Analysis Status Auto Save F C Andor iQD ata M Browne Revolution Time Lapse autolmages fai Acauisition Calibration Select eos 897_40xwi Image Window at e Protocol Type Time Series a Camera Binning Use Current t Camera Selection Use Current Events Pause Click OK to start protocol can if oe D Repeat T 20 times 0 ms fastest Acq u IS iti O n Repeat Channel CFP GFP AN Device Setu a 4 Channels
118. press the Set button Continue through uw us each channel The Plug in works on 8 12 16 bit grey and 32 bit float and 24 bit color or rev color images on the image disk It requires images with an explicit Z dimension which must have more than one Z plane The operation steps are explained for two different cases single channel image and multi channel image 366 Andor Technology Andor iQ USER GUIDE 6 9 1 Single Channel Image When using a single channel image with a time series follow these steps Select a single channel image from the image list select Best Z from the Edit Menu on the Image Window Use the Image Navigator at the bottom of the image window to set the Time Tab select the Z Tab on the navigator window Change the Z dimension until the best focused slice is found and press the Select Z button on the Dialogue box as shown in the figure below Best 7 psf_dtimes In this page You need to get the best focused z slice displayed in host application s viewer then click the Select 2 button This will record the selected lt position and will set the image to the next point When all points are done you can click Create to generate the selected Best image then Show to display the new image in viewer Time O Selected 21 Time 1 6 Clear All Close This will write a record in the Memo Box on the upper part of the Plug in window and advance the time di
119. rectangular background ROI on the image Use image frame number along operating dimension subtracts the appropriate image from each frame in a time series Typing the frame identification number into the text box specifies the frame required for the correction procedure iolx A separate image independent of the File out Source Background Mask Calibration time series Can also be selected using Numerator Backgroun f Use constant value of I the Use separate image option The Use maximum of area of interest number background image is selected from the Use image frame number along operating dimension i Use separate image Image List by clicking on the Select Mumerato SINETSERVERSDEMOIMAGES CA RATIOSFURA TIF Select i SSRIS button or by typing the designated file Denominator Backgroun Use constant value of name into the text box i Use maximum of area of interest number Use image frame number along operating dimension f Use separate image Denomata WINE TSERVERSOCEMOIMSGES C4 RATIOSFURA TIF Select Source Background Mask Calibration Ok Cancel Help Numerator Mone Threshold O Denominator Threshold 0 Andor Technology 386 Andor iQ USER GUIDE For the HEK cell example the second background correction method has been chosen for both the numerator and denominator In this case an ROI has been drawn in a background region of the image Now as each frame
120. red cross signifying that it is marked for deletion A second double click on the cell will restore it to the data set this will remove the cross tag from the image which will no longer be marked for deletion Binned histograms can be plotted for any of the available cell parameters To plota histogram click the left mouse button once over the tick box next to the parameter name so that a tick appears in the box and then press the Histogram button The histogram plot function can also be accessed from the menu bar at the top of the Image Review window 293 Andor Technology Andor iQ USER GUIDE File ames Data Histogram Sceatterplol The use of the histogram dialogue is described in Section 5 7 6 3 Note To plotthe histogram for a different cell parameter it is necessary to first click the left mouse button once over the first parameter tick box to remove the tick from the box similarly scatter plots to compare any two cell parameters can be plotted To plota scatter plot ticks must be present in the tick boxes next to the two parameter names before pressing the Scatterplot button The scatter plot function can also be accessed from the menu bar at the top of the Image Review window i Image Review ice Aletognann Scatherplot The use of the scatter plot dialogue is described in Section 5 7 6 4 The OK button exits the Show Spots dialogue first asking the user for confirmation to delete the
121. related actions and to Channel and Protocol wizards Further information on the use of the Wizards is described in Chapter 3 Andor iQ 2 2 Andor Camera File Image List Settings MORES Process Analysis Plug Ins Help D ai Calibration P a Stage Alignment a Select FRAPPA Device ap Current Open Imaris Select OSD Light Source Channels Acquisition Analysis RGE Status nalpsis Protocols DSO Calibration FRAPPA Calibration DSO Filter Change Camera Alignment Stage Registration Select XYZ Device 53 Andor Technology Andor iQ USER GUIDE 2 5 5 The Process Menu selecting Process from the file menu accesses additional functions These functions are listed in the adjacent picture A comprehensive description of the Process options currently available may be found in Section 5 1 2 5 6 The Analysis Menu Andor iQ 2 2 Andor Camera File Image List Settings Wizards Process EQ Re Plug Ins Help Region Histogram Yy a FRAP Analysis Depth Calculator of these functions is described Acquistion Analysis BGB Analysis MovisEd Tracker Line Frofile Further information on the use Current in Sections 4 6 Status 2 5 7 The Plug Ins Menu rece Help selecting Plug Ins from the file menu accesses i e additional functions fp RGB Merge Photobleaching Compensation The selected functions are now available for use during subsequent iQ sess
122. required position When the Save Histogram Data button is pressed the raw data corresponding to each time point is written to an Excel compatible data file The Print Graph button enables the displayed surface plot to be printed for further inspection and comparison Details of the save and print functions were described previously in this section 314 Andor Technology Andor iQ USER GUIDE 5 8 Kymograph Kymography Default Update Image Depth Dim Line Width Kymograph Line Profile Data Time 100 l E l 7 Display Mode Single Slice v nap a OC eee VE Tracking ONOI NE BS I Multi The Kymography Window is opened by selecting Kymograph from the View Menu at the top of the Image Window Two windows will appear as seen in the above picture The left side is the current image with a polyline and the right side is the actual Kymograph 5 8 1 Creating a Kymograph Once open a line or polyline must be drawn on the image using the appropriate button from the tool bar Press the Update Image to provide the most recent image Depth Dim Max Int Proj Update Image Min Int Pro Time 100 Create Display Mode 1e m select the Depth Dimension and Line Width from the corresponding drop down list Next Select the Display Mode from the dropdown list Once satisfied with the image 312 Andor Technology Andor iQ USER GUIDE press the Create button The status bar located under the
123. sF Hotfix RBS23980 Ensure that the Sentinel System Driver is listed If it is not repeat the installation process described previously 404 Andor Technology Andor iQ USER GUIDE There are no other devices connected to the parallel port sometimes having a printer or other device e g another dongle attached to the dongle can cause problems There is no other software which may interfere with the parallel port Similar to having a device connected The parallel port is enabled correctly in Device Manager and the BIOS Right click My Computer on the Desktop gt Properties gt Hardware gt Device Manager System Properties i System Restore Automatic Updates Remote General Computer Hame Hardware Advanced Add Hardware Wizard TF The Add Hardware Wizard helps you install hardware Add Hardware Wizard Device Manager The Device Manager lists all the hardware devices installed on your computer Use the Device Manager to change the properties of any device Hardware Profiles Hardware profiles provide a way for you to set up and store ai different hardware configurations Hardware Profiles OF Cancel Apply 400 Andor Technology Andor iQ USER GUIDE A Device Manager Miel File Acton iew Help gt Hig4 2 a cae Batteries Gl yf Computer Fee Disk drives m E Display adapters Bl DVD CO ROM drives Floppy disk controllers Gl
124. shown below The parameter to be plotted in the histogram is select Parameter for Histogram selected by clicking on the appropriate radio O Area Perimeter button Max Chord shape Clicking on the Cancel button exits the dialogue Integrated Intensity eon ene and returns to the Data Review panel Log Integrated Intensity Mean Intensityi4rea oe 6 Once the required parameter has been chosen click on the OK button to continue Andor Technology 296 Andor iQ USER GUIDE Time resolved data All Data Save Histogram Data C Animate Through Time A nimate Delay 0565 A Mex 3781 848 Bins o Frame D 2 Mean 1051 655 DaD Close C Statistical plot so a7 ani Update Display F kean Teo Cv 64 4 62 8 Get erate Mex M Min i Number Spots 102 57 Set Defaults Print graph C Surface Plot In the upper part of the panel a binned histogram of the chosen cell parameter is plotted Statistics are displayed below the histogram plot on the left of the dialogue for the complete histogram and for the main peak bin displayed on the histogram in red The minimum and maximum values to be displayed in the histogram can be specified in the Histogram Data section of the panel together with the number of bins in the histogram by typing appropriate values into the text boxes After changing one or more values click on the Update Display button to update
125. simple and consists of a slider Curent l n Live n Snap J Recora New r Camera Andor AMH Andor DSD Prior Lumen Andor APZ Contiol Settings range and is used during scan setup and manual MICRONS which sets the Piezo value in the configured operations The control dialog is shown below The readout shows the current setting and the buttons left and right of the text box below the lt lt lt 51 55 gt gt slider move the Piezo stage by increments of 0 1 and 0 5 um respectively 496 Andor Technology Andor iQ USER GUIDE A3 13 Generic TTL control from Andor iQ The hardware interface required for the Generic Shutter GS adapter is flexible according to the control requirements When you create and load a configuration with GS you select which interface to use as shown below GS offers 8 Line TTL Shutter using a PCI DIO24 card Appendix 3e Nikon LUSU and Uniblitz shutters via RS232 as well as 8 Line TTL control via Parallel Port LPT allona Windows PC Acquisition Auxiliary Devices a y o e EZ TRIT Live 5 nap Record 5 tay on T op Camera Generic Shutter Andor DSD Andor APZ hardware including shutters and The LPT parallel port has 8 TTL output lines for control of Output Set Up other hardware such as Status Not Connected Lumencor light engine EE perfusion control and so on The LPT1 interface is standard on LPT Pott Andor Imaging workstations D e
126. specified drive and then All Windows Logon Users will access a shared folder thus allowing all iQ users to share images Images in RAM specifies the number of frames that Andor iQ will attempt to keep in RAM at any one time Some image processing and visualization operations e g 3D View ClearView Kymograph RGB Merge amp Orthogonal View may require all frames to be loaded simultaneously therefore this setting must exceed the total number of frames being 430 Andor Technology Andor iQ USER GUIDE loaded The Default Value is setting is 65536 A2 1 2 Configurations Hardware Configurations can be created edited and deleted These functions are available through the Buttons on the Configuration Manager interface or through the File and Edit Menus Running the Configuration Manager executable file shows the panel illustrated below The available configurations are listed in a Tree View each comprising the following information ae Device Configuration Manager x File Edit Settings Toos About Exit Configuration Wizard New Edit Delete Exit Current User Mark Browne Current Configurations y Created By Mark Browne o Date Created 2 24 2005 9 28 01 AM o Date Last Modified 2 24 2005 9 28 01 AM 4 Acquisition Device Andor Camera a Auxiliary Device 1 Ludl Stage Device 2 Auxiliary Device 2 Ludl Filter Wheel Device 4 Auxiliary device OCX files used by this
127. system with at least 30GB or 30 free space Second fast hard drive e g SATA to make optimal use of ImageDisk In Andor supplied workstations we provide a pair of SATA hard drives in RAID O configuration to ensure sufficient bandwidth for streaming high speed imaging data This avoids limitations of system RAM or slow downs due to Windows file swapping e Graphics adapter with a minimum of 1280x1024 resolution and 16 bit color and a suitable display monitor Andor iQ has been designed to make use of dual monitor systems when provided We recommend at least a 19 monitor A sensitive CCD or EMCCD camera can be used e There must be either a vacant PCI slot for the imaging card Andor CCI 23 card or CCI 24 PCI Express will deliver optimum performance or alternatively iQ supports USB 2 0 cameras such as the Andor Clara and Luca series Some cameras running under Firewire IEEE1394a b are supported OHCI compliance is required e g Texas Instruments chipset required Other cameras supported by iQ have their own interface boards and these generally require a PCI slot e To run the full version of Andor iQ you will need either a license provided for evaluation or a software protection key dongle The Dongle will be of USB type We are always pleased to hear from users with information on their applications feature requests Suggestions and ideas Please feel free to drop us an email to the 10 Andor Technology And
128. the CSU imaging Channel focus the specimen in confocal mode and make 136 Andor Technology Andor iQ USER GUIDE sure that no vignetting is seen If there is an issue here repeat step 2 until this is resolved 3 Preliminary setup requirements a we assume that you have an iQ Configuration that includes all the devices you wish to use including the ALC with MPU selected camera and CSU b Make sure that you have a suitable sample for photo bleaching either Andor Test Slide 001 or a fixed Molecular Probes specimen e g Cells 2 where microtubules are labeled green excited at 488 nm c Start Andor iQ 1 8 1 or later exe dated Nov 15 07 or later d Make sure that FRAPPA is connected on a suitable COM port and let FRAPPA complete its self calibration the scanner goes through a self test a start up and when completed will show green lights on the controller front panel This takes about 30 seconds Note the FRAPPA calibration is independent of objective magnification but depends critically on the camera orientation alignment and pixel organization FRAPPA Selection FRAPPA Channels 488 FRAPPA lt lt Add 488 CSU chi Remove gt gt Remove All gt gt Reset FRAPPA Device e Create CSU Imaging Channel and a FRAPPA Bleaching Channel and then assign the FRAPPA Channel in iQ in the Select FRAPPA Device as shown in the figure above It is sensible to reduce the laser power to about 10 in FRAPPA chan
129. the LPT interfaces above If you have a PCU or a BOB device from Andor with free lines for control then include the Trigger and Event interface in the configuration The control interface to both adapters is similar so we use the Generic Shutter as the example here Acquisition Auxiliary Devices ag e E 2 Start iQ and you will see the uren ecor i Stap Top l ere Reemi JU Ser Generic Shutter in the Device Camera Generic Shutter Andor DSD Andor APZ Output Set Up setup dialog Unlock the dialog Status Connected On LPT1 control padlock and Select DA ler Skater OA 8 Line LPT Shutter and LPT1 or LPT2 Note LPT2 is used for LPT Pott LPT1 v PCI or PCle boards Then switch to the Output tab and setup TTL to LLE wavelengths LPT Connections as shown below Andor Technology Andor iQ How To Documents 3 When you switch to the output tab you will see the 8 outputs e r SiT Camera Generic Shutter Andor DSD Andor APZ labeled generically As shown on the right you can edit the Trigger Output Set up oO a a a a a Name and Actions The LLE logic a defaults to TTL Low for ON and States TTL High for OFF so you can Tlu ose Ax cs Open o rename High Low and High Low Fo E actions pulse actions UlLowHign Open Ciose O appropriately Set the Trigger paar w Name to the LLE channel color and or wavelength as shown Note there are two user defin
130. the following options Bakcground Select the option to apply to image _ Andor Technology 314 Andor iQ USER GUIDE mogra ine Profile Data 5 8 2 3 The Data Tab Kymograph Line Profile Data Profile Data Save Profile Data The Data Tab shows the Profile and Tracking das a a 22337 152 082 403 670 315 526 me 23 993 152 165 402839 319 664 24 990 152 247 403 020 313 955 25 966 152 329 404 657 320 986 26 983 152412 405 567 322 793 27 960 152 494 409 897 320 061 20 976 152 577 408 878 321 390 29 973 152 659 409 964 324 623 30 9639 152741 414 346 323 494 31 966 152824 411 294 320 625 11 32 363 152906 412418 320 151 12 33 959 152900 416843 320 927 Data The Save Profile Tracking Data button can be used to save the results in an XLD format to oo J 00 On ee oo ho oo i O XLD format is term we use for a text based x format where tabs are used as field separators in the data It can be easily read into Excel Origin Save Tracking Data Tracking Data JESUS and other spreadsheet and graphing tools me Sse 54900 omoa a S 1 1 56 516 154853 0 00 10 127 0 1 1 2 5 7147 154305 0 00 10 270 0 1 1 3 57 778 154 957 0 00 10 410 0 1 1 4 59 409 156 009 0 00 10 550 0 1 4 1 5 53 040 155 061 0 00 10 688 0 1 1 B 53 671 155 113 00 00 10 5456 0 1 1 F 60 302 155 165 0 00 11 029 0 1 Save in Y Calcium be 1 a 60 933 156 278 0 00 11 208 0 1 1 g 61 564 155 270 0 00 11 402 0 1 1 10 6
131. their Protocols private This can be done by making a suitable configuration for each user include the user s name in the configuration In this way you can allow multiple users to control the same hardware but maintain independent Protocols Scans and Channels setup vvizard Protocals The Protocol Manager is accessed Protocol Manager by clicking on the Protocol button on the Acquisition Page or from the Pl Wizards menu A Protocol contains a Delete Copy select Cancel Protocol Summaries series of keywords which define Actions Current Protocol Newtest New Protocol To create a new protocol is click Select name tor new Protocol H Protocol Bad 49 Protocol Ne Multiphase MCZT New Enter a name to identify the Protocol 429 Protocol 429 Protocol 429 Protocol 429 Protocol 429 Protocol 429 Protocol 429 Protocol 429 Protocol Protocol aw m i Test Case 10 T L MC MF Z Test Case 11 MC MF Mon AF Mon Test Case 12 MC MF AF Z Test Case 2 Multi Phase vvith Repe Test Case 3 Multi Pos one with z ste Test Case 42 repeatlevel Frappa Test Case 5 Burst within Z Test Case b Multi Phase with Autor Test Case 7 Z First Test Case O Channel First ho I pie re E HT Protocol non aK Cancel protocol A prototype Protocol is then Be inant tal NS f Protocol Phase created and it can be edited in the
132. time_shutter1 5us_8bit_1280x102 E time_shutter 5us_Sbit_1280x1024 seq003 E time_shutter1 5us_Sbit_1280x102 a time shutter Sus _Sbit_1280 1024 seq004 Fa time_shutter1 5us_Sbit_1280x102 Fa time_shutter1 5us_8bit_1280x1024 seq005 Ea time shutterl5us 8bit 1280102 Es time_shutter 5us_Sbit_1280x1024 seq006 E time shutterlS5us Bbit 1280X102 i File name time_shuttert Sus_Sbit_1280 1024 seqQ01 Files of type TWICE Ts i ka a Cancel selecting the first file in the appropriate directory and clicking on the Open button loads a group of individual images constituting a time series For further details about opening images please refer to Sections 2 9 1 and 2 6 1 5 7 3 Using Spot Analysis Clicking on the Spot Analysis tab in the iQ window accesses the Spot Analysis module A successful analysis can only be performed following the definition of a suitable experimental protocol Analysis AGE Analysis Calibration Movie Editor spot Analysis Manage Analyse Review Il 977 Andor Technology Andor iQ USER GUIDE 5 7 4 Managing Analysis Settings Manage Analyse Review The Manage dialogue is used to set up an analysis protocol and specify the parameters for analysis The set up procedure is accomplished with the aid of a set up wizard and defines the threshold and filter limits for the analysis A new analysis protocol file can be created or an existing protocol file edited during the set up procedure
133. to perform a manual copy of two folders as detailed below You can perform these steps while iQ 2 IS running 28 Andor Technology Andor iQ USER GUIDE Step 1 Before you start iQ 2 your Windows system hard drive will look like this in C Documents and Settings All Users Application Data Kinetic Imaging iQ folder File Edit View Favorites Tools Help ae O Back J wi Search Wey Folders Bek Address C C Documents and Settings 4ll Users Application Data Kinetic Imagingli ha Go x Folders E all Users A Durnrny E O Application Data Adobe CS Andor 9 Andor Bioimaging Apple Ee Apple Computer 2 FLEXnet C Google CO Intelligent Imaging Innovations Lastsessioninlo oh Kinetic Imaging A Settings 3 Common C3 Configuration LastSession rgn Fou pro RGN File 3 Dummy dn 3 No Grab 9 Revolution Time Lapse C Microsoft gt Research In Motion C SalesLogix Seagate C Skype Revolution Time Lapse After you start iQ 2 a new folder called iQx is created which will store new configuration data for Protocols and Channels As shown below Step 2 After you have checked that the relevant folder in this case iQx Revolution Time Lapse has been created you can copy all of the files from folder iQ Revolution Time Lapse to iQx Revolution Time Lapse Now when you look in the iQ 2 Protocol Manager and Channel Manager all of the Protocols and Channels will hav
134. to the PE2 rear panel You will see the PE2 LEDs light up as the camera exposes to capture a frame e Luca R can produce an erratic display of flashes indicating that the Fire signal is reset when frames are demanded so you may observe more than one flash per frame Pulse mode operation ensures the LEDS are on only during camera exposure and therefore minimizes specimen exposure rather like the Active Blanking Feature we use in the laser combiner In Pulse mode the PE2 is configured to accept TTL pulses from the camera Fire signal to gate the LEDs on and off via input pin 9 and pin 1 Gnd of its 15 way sub D connector on the rear port of the device A special cable is available from Andor to couple SMB to D15 and if required to Direct TTL control for maximum speed Pinout for the TTL cabling solution is shown in the next section 489 Andor Technology Andor iQ USER GUIDE Table 3 12 1 Connections for the iQ TTL Pulse mode cable for the CoolILED are as follows Parallel TTL Port The precisExcite is fitted with a 15 way D type socket to allow external TIL signals or switch contacts to control the switching on and off of the LED arrays 0 o 0 0 01000 O O Os O O Op O Front View of D type Socket TIL Drive Switch Contact Drive Channel 1 TTL Input Pin 1O ve Pin 3 Gnd Internal 5V Channel 2 TTL Input Pin 11 ve Pin 4 Gnd Channel 1 Switch Input Pin 10 Pin 14 Channel 3 TTL Input Pin 12 ve Pin
135. toad Crop size and shape Regions Sove _Cycle Fraa IO can be of line polyline Edit Classification s Set Reference rectangular or elliptical in type and more complex figures can be drawn Time z Wavelength 0 00 00 000 heveme FrameNumber Mintange Maxrange increment a intervaliems a a co ad aliaj alpi ojo Ti olan Sein semaj freehand polygon tools w DE Automap 141 190578 644 146 using the polygon or The analysis of ROls is useful for the through series analysis of temporal or Z series data Discrete regions drawn using the same color can be classified and assigned to a particular feature or class assigned by the user Multiple regions from the same class may be analyzed individually or combined as a group and a background correction can be applied during analysis using background ROls Further information regarding ROI classification and the use of the Feature Definition function is described in Section 2 8 4 The use of ROIs during image analysis is discussed in Section 4 6 1 39 Andor Technology Andor iQ USER GUIDE 2 4 Supported Image File Formats Andor s file handling tools allow for import of wide a range of file formats as described subsequently in Section 2 5 1 Imported file formats supported by iQ are listed as follows AVI BioradMRC Bitmaps Fenestra Kinetic Imaging Olympus GB200 JPEG jpg jJoeg Kontron IMG Micro Voxel MIF Multi Load Phot
136. using the Analysis Manager wizard When the Manage tab is selected the following panel appears Analysis RGB Analysis Calibration Movie Editor Spot Analysis T h e exam p e p ane S h ows a st of Manage Analyse Review experimental protocol files created during New Edit Delete previous Spot Analysis sessions Current analysis Carbon12Bit Spot Analysis Carbon 2Bit Spot protocol files are selected by clicking 3 Spot Analysis Carbon 6Bit H 8 Spot Analysis Carbon32Bit Hg Spot Analysis Carbon8Bit H Spot Analysis Optilas1 2Bit 8 4 Spot Analysis Optilas8Bit symbol reveals the analysis setup details on the relevant file name Clicking the for a particular protocol The listing shows the image used to define the protocol whether bright or dark objects are to be measured the thresholds used to create a binary image for subsequent measurement and the filter criteria used for data exclusion 278 Andor Technology Andor iQ USER GUIDE Analysis RGB Analysis Calibration Movie Editor Spot Analysis A n exam p e p ane S h owi n g t h e Manage Analyse Review experimental details for the protocol file co Eat Delete Colona is shown left Current analysis Colona lS Spot Analysis Carboni 2Bit y Spot Analysis Carbon 6Bit x Spot Analysis Carbon32Bit 5 Spot Analysis Carbon Bit Sree Spot Analysis Colona Setup Image Image deleted
137. x Camera Trigger Camera Trigger Input Output Set Up Input Output Set Up o ay a A a a o EEES Ea EO p Trigger Name no Trigger Name Out0 States States Thh Fih o Shs Ts Low Thews R TL hich tow HighLow Low Event a Jon Low High LowHiah Transition Delay fo ms Interval fig SX 5 Depending on which TTL signals you intend to use O thru 7 for Input and Output you can monitor or set their status on these tabs The 8 buttons along the top of each tab switch the user interface to reflect the current state of the relevant inputs and outputs Inputs sense TTL levels so that input signals must remain in the defined state long enough for iQ to sense while other actions are taking place this depends on other actions but a minimum of 100ms is a good starting point Outputs are used to send TTL levels or pulses as indicated above On the Output Tab Transition delay is measure of the response time of an external device if it needs to follow the Output state Interval defines for how long the pulse state will be held E g 1 if you want to send a pulse of 10 ms duration select the pulse polarity set Interval 10 ms Transition delay O E g 2 if you want to open an external shutter for 50 ms and it takes 15 ms to open and close fully then set Transition Delay 15 ms and Interval 50 ms 555 Andor Technology Andor iQ How To Documents 6 Now we are ready to Acquisition
138. you do NOT enable FLZ for Stage Alignment If you are using the side port on an inverted microscope you may need to physically rotate the camera by 90 degrees If that is not possible then you can use this software rotation step but it does have a speed overhead so only use it if you cannot rotate the camera This requirement results from the fact that the side port sees X motion of the stage in its Y plane and vice versa These precautions will make the system feel more comfortable to use and avoid confusion or errors in scan patterns Access to the camera orientation controls is via the Device Setup dialog on the camera tab 125 Andor Technology Andor iQ USER GUIDE Andor iQ 1 9 1 Mail 7 File ImageList Settings QC Wizards Process Analysis 15 x Plug Ins Help Setup Wizard Camera Orientation Multi Well Plate and Petri Dish Next gt gt Cancel Camera Orientation Ifa Multivwvell Plate or Petri Dish is to be used then the orientation of the camera is important 1 Use the stage to move of feature in the stage x direction Rotate the camera so that the feature aligns to the image window x direction 2 Please select Multi Well if you intend to scan multi well plates M Multi Vell Ae cri Beg ans si so Current Channel te st v Select Channel for viewing If no channel exists or needs to be edited close this dialog create or edit the channel and retur
139. 0 Fields 32768 112 500000 Fields 32768 112 500000 Fields 32768 112 500000 Fields 32768 112 500000 Fields 32768 112 500000 Fields 32768 112 500000 15312 296392 15435 082695 15499 316000 15435 082695 15686 335608 15435 082695 15312 296392 15636 097000 15499 316000 15636 097000 15686 335608 15636 097000 15312 296392 15837 111305 15499 316000 15837 111305 15686 335608 15837 111305 Region Info Fields End Environment User iQ User Configuration iMic Luca Environment End Protocol Description Protocol TL 3Ch MF Image TL 3Ch MF Protocol Type Time Series Camera Binning Use Current Wait Allocation Off Camera Selection Use Current Events Keyboard Events On Default name Spacebar Andor Technology Andor iQ USER GUIDE Scan 3MFMontageZScan Montage Acquisition XYZ Centre on Registration XYZ Pause Click OK to start protocol Interval O ms fastest Repeat 1 times End Protocol Description End Grab Parameters 525 Repeat XY Repeat Montage Repeat Z Z 54 18 um in 10 planes Montage Positions 9 3 by 3 XY Positions 2 XY 1 16168 142 16935 022 116 66 XY 2 15499 316 15636 097 112 5 Channel Current Camera EM Gain Current Camera Binning X Current Camera Binning Y Current Camera Exposure Time s Current LUT Current Map Min Current Map Max Current iMIC Filter Wheel
140. 0 1 10 1 Andor Camera With Fast Z and Wavelength ES File ImageList Settings QC Wizards Process Analysis Plug Ins Help apparent how to adjust these settings for your User Andor Configuration FLZ Trigger own specific Image requirements ImageType 16 bit grey pseudo Image Size 512 x 512 Number of Planes 1000 14 Please contact Andor xY Cabbeation WOES technical support team if you have further Acquisition Analysis AGB Analysis Calibration Movie Editor Spot Analysis questions or Status suggestions Auto Say M G Andor iGDataAndor FLZ Trigger Autol mages aa Image Test Fast Trigger Protocol Type Fast Time a Camera Binning Use Current a Wait Allocation On SQ Camera Selection Use Current Pause Click OK to start protocol Interval O ms fastest E Repeat 10 times A Trigger In InO High Device Setup Trigger In Ind Low Move Current Loop Protocol E Channel Current MV Display Image Snap no Trigger Out OUtO HighLow End Channel 558 Andor Technology Andor iQ How To Documents A5 18 How to Debug DSD Calibration Andor internal lf DSD fails to run the calibration procedure there could be several reasons but a common cause is the filter turret dichroics are slightly misaligned This can result in offset in CCD camera images Try Calibration several times to be sure that a repeatable problem exists If you need to debu
141. 0 12 27 05 db a Acquistion Device Andor Camera the Screen shot below Make sure that the Global settings are configured as shown below so that the ImageDisk uses a suitably fast hard drive for streaming In this example a pair of high performance 1GB 7200 rom SATA disk drives configured in RAIDO mode were Image Disk Configuration ImageDisk Path Virtual Image Disk Default Images are NOT retained after shut down Recommended for Komet Users Select a Drive if you want Images to be retained after shut down Recommended for iQ Users An ImageDisk folder will be created on this drive Important An NTFS format hard disk drive which does NOT host the Windows Operating System will improve performance Total Disk Space 1863 01 GB Free Disk Space 1861 66 GB D Data selected as drive D Data as shown below 222 Andor Technology Andor iQ USER GUIDE 4 7 2 2 Starting two instances of iQ When you have the optical setup configured then you will need to choose which camera will be loaded by the Master configuration i e FLZ We refer to FLZ as the Master instance because it is FLZ that controls the wavelength Z and trigger sequence of the cameras The choice is arbitrary but iQ will remember for FLZ sessions Start iQ select the FLZ configuration and then the dialog below will be shown Camera Selection The camera selected using the drop down list and is identified by serial number which is printed
142. 00 times O ms fastest EG Repeat Channel BFP_GFP lo Channel Description Sed 2 nap Frame i Ss End Channel of End 11 Lise a End T 3 Click Protocol and reselect MyTemplate this will make sure that the protocol files are saved to disk 4 Using explorer navigate to C Documents and Settings All Users Application Data Kinetic Imaging iQx or the equivalent for your installation of Windows 5 Select the folder with the name of the current configuration i e iXon Prior IX81 Piezo ALC Frappa see image 568 Andor Technology 569 Andor iQ How To Documents File Edit View Favorites Tools Help Q ex X gt y y Jo search gt Folders ES x e Ea Address E G Documents and Settings 4ll Users Application Data Kinetic Imaging iQx Go Folders xX Names SYT Size Type se Win XP G Dummy File Folder Acronis Disk Partition riz File Folder i O Andor Gixon File Folder El 3 Documents and Settings ixon ALC Trigger File Folder i E 3 All Users Gixon IX61 ALC Piezo FRAPPA Prior File Folder El B Application Data Gixon IX81 ALC Piezo Prior Frappa File Folder Acronis CFixon Micropoint File Folder 32 Andor Gixon Prior X61 Piezo ALC File Folder B Andor Bioimaging ixon Prior IXS1 Piezo ALC Frappa File Folder 9 AutomatedQA Gixon TI ALC Piezo File Folder i Borland No Grab File Folder E Carl Zeiss Test 2 iXon 1X81 ALC FRAPPA Prior Piezo File Folder E Kinetic Imaging Tes
143. 0000 lux x 1392 0 161250 um y 1040 0 161250 um PERG to rego List 6th Dim A i WELL 3 MONTAGE TALIN BS PHALLOID 1 8 bit grey MONTAGE KOHLER JPG_Rotate90 2 24 bit col MONTAGE KOHLER JPG e 24 bit col CAGEDCAMP_1PORUV_5MS_4x4 0000 TIF HiT E 16 bit gre MFMONZT 2CH 2 2 16102009 TIF_Montage 5 16 10 2009 14 bit gre Wavelength 2 5 ey 5 Time 3 MFMONZT 2CH 2X2 16102009 TIF 6 16 10 2009 14 bit gre Wavelength 2 5 Montag Y 5 TOPRIGHTALIGNMON TIF 7 22 06 2006 12 bit gre Time 1 Exposur PR Pe FFT Lee met rie Close Refresh Show Rename Image Save Image Delete Images Image Details When an image is selected in the Image List the meta data information about the image is displayed here Details of the content and layout of the Image meta data are included in Appendix 4 of this user guide lf more than one image is currently selected details are displayed of the latest one to be selected Image List click on an image name to select Selected images are highlighted You can use standard multi select key strokes click with CTRL and Shift to select more than one image You can Delete or Save the single or multi select with the appropriate button click 47 Andor Technology Andor iQ USER GUIDE Most buttons are self explanatory but where more information is needed we provide it below Refresh this button refreshes the Image Manager interface Function key 5 F5 w
144. 09 If Self Operation taking average of selected range for secondary is selected Operation Select the Operation from the dropdown list as follows lf Subtract is selected as above picture shows from the igi Modulus dropdown list an additional box will appear giving the user the following options Operation Add lf subtract is not selected no other selections will be necessary 401 Andor Technology Andor iQ USER GUIDE Diadic Source Self Operate Diadic Source Sell Operate A Use the Select button to Ruming UM III elec select an image from the image list Region of Interest B When Time is selected use Region Of Interest B i the dropdown menu to select f Time a Start and End Value For secondary average images Start 177 End 509 6 12 2 The Background Tab Source Background Mask Background 4 fe Use constant value of f Use maximum of area of interest number C Use image frame number along operating dimension f Use separate image Background B f Use constant value of f Use maximum of area of interest number f Use image frame number along operating dimension f Use separate image B pr Ee Te TT f Use maximum of area of interest C Use mean of area of interest 402 Andor Technology Andor iQ USER GUIDE Background A Background B This tab is used to specify the Background Correction Values which will operate before the primary
145. 1 Current iMIC Filter Wheel 2 Current iMIC LED Shutter Current iMIC Piezo Z Motor Current iMIC TIRF Angle Current Snap End Z End Montage End XY Andor Technology Andor iQ USER GUIDE 926 Camera Andor Window Left 0 Window Top 0 Window Right 1003 Window Bottom 1001 Window Width 1004 Window Height 1002 BinningX 1 BinningY 1 Image Width 1004 Image Height 1002 Exposure 20000 Grab Parameters End Tab Device Info Camera Luc285_MONO 574 ControllerCard DriverVersion 2 86 30001 0 CofVersion 0 VxDVersion 0 0 SoftwareT rigger Supported BitDepth 14 FPGA_Version 16 1 Ocx_Version 5 1 1 19 Ocx_Time 15 08 50 Dec 10 2009 Ocx_File C PROGRA 1 ANDORB 1 COMMON 1 KIDAND 1 0CX Tab Device Info End Tab Exposure Window 1004 1002 Shutter Open Binning 1x1 Temperature Actual 20 Display Control Fastest Checked Display Control Display Rate 1 Gains Pre Amp Gain 1 00 Andor Technology Andor iQ USER GUIDE EM Gain Enabled Unchecked Gains EMGain 21 Exposure Time Frame Transfer Unchecked Exposure Time unit ms Exposure Time exposure value 20 00 Exposure Time Actual 20 000ms Exposure Time Readout in ms 80 644 Experiment Control FLZ Unchecked Experiment Control Trig Mode Free Run Experiment Control Ext Start Unchecked Use Frame averaging Unchecked Frames to be averaged 1 Fastest Grab Unchecked Available memory 1459 Tab Exposure End Tab Temperature
146. 1 _Reference_1 13 255 Selected All Period Data for selected points Update Point Data Table Period Channel Start ms Start Ya End ms Endal Fit Start FitAsym Mobile Half Life Diffusio 0 0 103 00 36 426 994 00 59 785 36 426 58 189 0 64268 43 631 0 88596 Note the following Each selected position in the Z dimension corresponds to one region consisting of the 100 pre bleach frames and 1100 post bleach frames e The curves o Black Real data In this case the FRAP and reference data o Green Corrected data o Pink Fitted curves 5 13 3 Region Setup A region is defined by its Point Position its ROI and whether it s FRAP data or correction data ro FRAP Analysis C DOCUMENTS AND SE Mobility Chart xX Axis Mode Pre Bleach Correction To add a region Point z 1 Choose the point position ral a E EE Region Selection 2 Click the add button x FRAPPA Reference 3 The following dialogue will appear Choose th Farr a aeee UU click Add _FRAPPA_1 _Reference_2 336 Andor Technology Andor iQ USER GUIDE Add Region Available Regions 1 Please select the FRAPPA region to add _FRAPPA_1 4 The next step will list the reference regions Either add one or choose None as required The setup process will depend on what correction is being applied The user may choose one of the following correction methods No correction Selected by not choosing a refer
147. 2 195 155 322 0 00 11 569 0 1 yy 1 11 62 826 155374 0 00 11 729 0 1 1 12 63 457 156 426 0 00 11 889 0 1 A lt i gt File name Save as type XLD Files v Cancel 5 8 3 Lamella Mode in the Kymograph Plugin Lamella mode operation is aimed at quantitative analysis of lamella protrusion and contraction observed in membrane ruffling which is common in motile cells It s development was suggested by a number of users and is based on the work Hinz et al 1999 But Kymography in general can be used in a number of different scenarios for quantification and visualization of movements wavefront development crystallization and so forth 315 Andor Technology Andor iQ USER GUIDE 5 8 3 1 Step by Step Use of Lamella Mode 1 Load or show an image from the ImageList into iQ s Image Window The image must be a series with a dimension in which analysis is possible e g Time or Z or even spectra can be analysed In this case let is assume time is of interest as in lamella dynamics In the work of Hinz et al phase contrast images were used but fluorescence can also provide a suitable image data 2 Start the Kymograph tool 3 At the top right of the Kymograph window select from the Kymograph Mode combo box Lamella as indicated by the arrow in the figure below on the left Kymography E Eymograph Mode Depth Dim Update Image Timel75 4 At the top middle of the Kymograph window select
148. 21187 07 15410 587 112 34 XY 4 21661 07 15410 587 112 34 XY 5 22135 07 15410 587 112 34 Andor iQ USER GUIDE 517 Well 7 Well 8 Well 11 Well 12 Well 13 Well 14 Andor Technology XY 1 8753 683 17516 214 133 16 XY 2 9227 683 17516 214 133 16 XY 3 8753 683 18223 214 133 16 XY 4 9227 683 18223 214 133 16 XY 5 9701 683 18223 214 133 16 XY 1 11252 897 17453 532 133 16 XY 2 11726 897 17453 532 133 16 XY 3 11252 897 18160 532 133 16 XY 4 11726 897 18160 532 133 16 XY 5 12200 897 18160 532 133 16 XY 1 18750 539 17265 484 120 67 XY 2 19224 539 17265 484 120 67 XY 3 18750 539 17972 484 120 67 XY 4 19224 539 17972 484 120 67 XY 5 19698 539 17972 484 120 67 XY 1 21249 753 17202 801 120 67 XY 2 21723 753 17202 801 116 5 XY 3 21249 753 17909 801 116 5 XY 4 21723 753 17909 801 116 5 XY 5 22197 753 17909 801 116 5 XY 1 8816 365 20015 428 137 33 XY 2 9290 365 20015 428 137 33 XY 3 8816 365 20722 428 137 33 XY 4 9290 365 20722 428 137 33 XY 5 9764 365 20722 428 141 5 XY 1 11315 579 19952 746 137 33 XY 2 11789 579 19952 746 137 33 XY 3 11315 579 20659 746 137 33 XY 4 11789 579 20659 746 137 33 XY 5 12263 579 20659 746 137 33 Andor iQ USER GUIDE 518 Well 17 Well 18 Well 19 Well 2O Well 21 Well 22 O Andor Technology XY 1 18813 221 19764 698
149. 3 2 Channels Channels are settings files created and managed in the Channel Manager Channels specify all the settings for capturing an image of a specimen in a given contrast mode or with a specific fluorescence probe e g phase contrast or DAPI or TRITC Channels describe how your hardware devices are set up to illuminate control devices to the sample and capture the image They are set up using the Channel Manager which is opened by pressing the Channel button on the Acquisition dialogue Channels also control light source Shutter actions before and after taking an image and in order for this to be defined correctly you must set the shutters in the Channel Manager wizard as you will see in Chapter 4 Once a channel is configured through the Channel Manager shown here you can use it to snap single frame images share it with other users and build it into more complex acquisition sequences File Image List Settings QC Wizards Plug Ins Help DNA nn Protocols Channels specify settings for Channels Cameras microscopes filter wheels and Channel Manager shutters in short anything affecting sample illumination and image capture New Edit Copy Delete Close Channel Summaries Current Channel Channel 1 To create a new channel press New A BED Channel Channel 1 Ci Camera Brightness 128 ve Camera Contrast 128 eo Camera Binning 1 ow Camera Binning 1 AS Camera Exposure Ti
150. 353 fas 1979 467 0608 e mail intak ineticimaging com e mail info us kineticimaging com wy andorig com A3 2 2 Function Tabs Device Info Settings A3 2 2 1 The Device Info Tab p Camera Information PCE Version 0 EPROM Version O Selecting the Camera Tab will open Cof file Version 155 the Camera Settings Selecting the YxD Version 413 Driver Version Z fi Device Info tab will provide an Resolution 512x512 Bit Depth 14 overview for the camera being used Max Binning S 448 Andor Technology Andor iQ USER GUIDE A3 2 2 2 The Settings Tab Acquisition Auxiliary Devices l x The Settings Tab consists of two additional tabs the Exposure and Samea Prio DUSS _BY 3771 Settings neral Tabs Ge era abs Exposure General Device Info EI Sub Window 1 Real EM Gain The Exposure Tab allows you to modify Window 512 512 Enabled Binning 3 camera settings that pertain to iv xer EJ E 1x1 v experiments See Section A3 2 2 2 1 ps MShutter Pre Amp Gain 5 10 for more information No Intemal Shutter Pre mpGain Frames to be averaged 7 jeer Time 1 Frame Transfer a oa 30 0 fms Display Contro ui EH Display Every Frame General Tab contains settings that are C Display 5 Frames s Resio mel 32034 Actual Frame Rate 0 00 Specific to Andor Cameras These Free Run Ext Start
151. 37 14954 3174468164 124 83 908 11664 2140559637 14954 3174468164 95 67 11664 2140559637 15661 3174468164 95 67 13689 4281119273 14891 6348936329 120 67 13689 4281 119273 15598 6348936329 120 67 14637 4281119273 15598 6348936329 120 67 16662 642167891 14828 9523404493 116 5 16662 642167891 15535 8523404493 119 32 18687 8562238547 14766 2697872658 116 5 18687 8562238547 15473 2697872658 116 5 19635 8562238547 15473 2697872658 116 5 21661 0702798184 14703 5872340822 112 34 21661 0702798184 15410 5872340822 112 34 8753 682553 18356 17516 2140559637 133 17 8753 682553 18356 18223 2140559637 133 17 11190 2140559637 15661 3174468164 95 67 12138 2140559637 15661 3174468164 95 67 14163 4281119273 14891 6348936329 120 67 14163 4281119273 15598 6348936329 120 67 16188 642167891 14828 9523404493 104 16188 642167891 15535 8523404493 120 67 17136 642167891 15535 8523404493 87 34 19161 8562238547 14766 2697872658 116 5 19161 8562238547 15473 2697872658 116 5 21187 0702798184 14703 5872340822 112 34 21187 0702798184 15410 5872340822 112 34 22135 0702798184 15410 5872340822 112 34 9227 68255318356 17516 2140559637 133 17 9227 68255318356 18223 2140559637 133 17 Andor Technology Andor iQ USER GUIDE 909 ZScan Montage 9701 68255318356 18223 2140559637 133 17 11726 8966091472 17453 5315027801 133 17 11726 8966091472 18160 5315027801 133 17 18750 5387770383 17265 4838432294 120 67 18750 5387770383 17972 4838432294 120 67 19
152. 49 117 22201 229 129 78 XY 3 21375 107 22908 229 137 33 XY 4 21849 117 22908 229 137 33 XY 5 22323 117 22908 229 137 33 Channel Current Snap Camera EM Gain Current Camera Binning X Current Camera Binning Y Current Camera Exposure Time s Current LUT Current Map Min Current Map Max Current iMIC Filter Wheel 1 Current iMIC Filter Wheel 2 Current iMIC LED Shutter Current iMIC Piezo Z Motor Current iMIC TIRF Angle Current Andor Technology Andor iQ USER GUIDE 920 Window Bottom 1001 Window Width 1004 Window Height 1002 BinningX 1 BinningY 1 Image Width 1004 Image Height 1002 Exposure 20000 Grab Parameters End Tab Device Info Camera Luc285 MONO 574 ControllerCard DriverVersion 2 86 30001 0 CofVersion 0 VxDVersion 0 0 Software Trigger Supported BitDepth 14 FPGA_Version 16 1 Ocx_Version 5 1 1 19 Ocx_Time 15 08 50 Dec 10 2009 Ocx_File C PROGRA 1 ANDORB 1 COMMON 1 KIDAND 1 OCX Tab Device Info End Tab Exposure Window 1004 1002 Shutter Open Binning 1x1 Temperature Actual 20 Display Control Fastest Checked Display Control Display Rate 1 Gains Pre Amp Gain 1 00 EM Gain Enabled Unchecked Gains EMGain 21 Exposure Time Frame Transfer Unchecked Exposure Time unit ms Exposure Time exposure value 20 00 Andor Technology Andor iQ USER GUIDE 521 Exposure Time Actual 20 000ms Exposure Time Readout in ms 80 644
153. 55 43 SCAMS andthe SCAM Wizard assores DA LEN RR La DRA E LES TRA USAN LEA DRA BA ST EIS A ian 160 4 4 Protocol Tree Actions Insert Delete and Edit ccccccssscccsscccsscccsscccscccccsccccsccccscccsccccsccccscccosescess 190 4 5 Protocols WASICS sivcscncsescssscsiscssnasavassessdvcasncsacssnscadscasnasacsanecadesasnadassnscadscoanasedsanscadesossadesssnscadscsssasessaasasdcssssss 203 4 6 Flexible Protocol Patterns isscsscccacecssetasereancetecatoca teseaecasecstetaseresncetecatoaatexeancasacetetasersancetecsdaaadexsancasacetetaseseanss 210 AT NEG CaMer as ACGUISHION TA AA TA ATA E O dadas 219 4 8 Precision Control Unit PCU Triggers and Events c cccccsssssssscccsssssssssscccccccsssssssccccscesssssssscsseseess 228 5 ANALYSIS VISUALIZATION asaan aaaea aa DU aa au ad dota eU DNA AA O elude 237 Sel Imago6 Contrast and US dA ATA eve eE raaa EEE ENO 238 5 2 The Online Grap lia A aa Ea A aa A a DS E DE aaae 244 5 3 Thro gh Series A Mal YSIS crossroads inean enaena oranana aaae aaa asa Nde aean eaaa aa aari eiaa eeina aisi 246 5 4 RGB Analysis and Co Localization eeeeccccocccssssssessecececccoccososssssesecceccecosooosssssssesecececsosssosssssssscseceeesssss 252 SS ANALVSIS O PIONS asiaa a a aaa a a a aaa 261 S The Movie Editor Sroa A Aa AU aE aN A A RU DA SAA Aa EE EE 269 5 7 Fhe SPOE UNCHUON snai anaa aaaea e ea AEN ara a a 274 IO ACV IO BADIA oea EOE Eae TE S 312 5 9 OLUNOSONAL SCE Vie Wssisaeisisor oianean aei a
154. 6 000 50 000 0 575 116156 000 92 114 11 663 Cell63 15 523 000 110 000 34 000 0 543 36507 000 69 803 10 505 Cell64 15 758 000 130 000 39 000 0 564 66491 000 87 719 11 105 Cell65 15 749 000 134 000 44 000 0 524 57678 000 77 007 10 963 O cell66 15 1384 000 198 000 63 000 0 444 133665 000 96 579 11 803 Cell6 15 1166 000 158 000 45 000 0 587 119869 000 102 804 11 694 Histogram Cell68 16 903 000 148 000 43 000 0 518 6315 000 84 513 11 243 Cell69 16 698 000 120 000 34 000 0 609 63339 000 90 744 11 056 Cell 70 16 1052 000 152 000 50 000 0 572 103338 000 98 230 11 546 Cell 71 16 779 000 130 000 37 000 0 579 36473 000 46 820 10 504 Cell 72 16 823 000 130 000 36 000 0 612 67205 000 81 659 11 116 Cell 73 16 760 000 130 000 39 000 0 565 64076 000 84 311 11 068 O Cel 74 16 3504 000 300 000 84 000 0 489 519853 000 148 360 13 161 X Cell 751A 848 nnn 14 nnn 44 Nn NAA ZSA7O NON AA AAR 11 238 of Total Spots 102 Histogram Scatterplot Save Data Clear Selection X Cancel Included Spots 99 All Data Single Frame Clear Selection ignores the selected cells and Cancel returns you to the Data Review dialogue panel The OK button exits the Show Data panel prompting the user to confirm that the rejected features should be deleted Once a feature and its data have been deleted it cannot be restored so make certain that the selection is correct before pressing the OK button To exit without deleting any cells simply press the Cancel button 290
155. 66 Andor Technology Andor iQ USER GUIDE be visible below the Image Toolbar This tab allows you to switch the display between the Ratio and the Raw images The Image Toolbar Regions of Interest ROI s can be defined about features of interest on the image using the Define Point Define Line Define PolyLine Freehand Polyline Rectangle Ellipse Polygon and Freehand Polygon buttons and classified according to user specified features using the Current ROI button in the Image Toolbar Section 2 10 17 Statistics can then be calculated for each ROI or class of ROI in the Analysis or RGB Analysis dialogue 2 1 Image Contrast or Mapping Control EU The Manual Mapping button opens the Image Contrast dialogue which presents histogram s and tools for setting the Maximum and Minimum mapping values This is shown on the next page The mapping transfer function is shown as a red line overlaying the histogram plot For multi channel images one set of tools is provided per channel Changing the Max and Min values and adjusting the transfer function will modify the display in the Image Window to improve contrast or view specific image features The underlying image pixel intensities remain unchanged Options View lt lt EE E 4291 000 Max M Invert ES 1067 000 Min Exclude __ a 00000 step Gamma Coe
156. 698 5387770383 17972 4838432294 120 67 21723 7528330019 17202 8012900459 116 5 21723 7528330019 17909 8012900459 116 5 8816 36510636712 20015 4281119273 137 33 8816 36510636712 20722 4281119273 137 33 9764 36510636712 20722 4281119273 141 5 11789 5791623308 19952 7455587438 137 33 11789 5791623308 20659 7455587438 137 33 18813 2213302218 19764 6978991931 129 18813 2213302218 20471 6978991931 129 19761 2213302218 20471 6978991931 129 21786 4353861855 19702 0153460095 124 83 21786 4353861855 20409 0153460095 124 83 8879 04765955069 22514 642167891 124 83 8879 04765955069 23221 642167891 129 9827 04765955069 23221 642167891 129 11852 2617155144 22451 9596147075 120 67 11852 2617155144 23158 9596147075 145 67 13877 475771478 22389 2770615239 112 34 13877 475771478 23096 2770615239 116 5 14825 475771478 23096 2770615239 112 34 16850 689827441 7 22326 5945083403 108 17 16850 689827441 7 23033 5945083403 108 17 18875 8038834054 22263 9119551568 104 18875 8038834054 22970 9119551568 104 19823 9038834054 22970 9119551568 104 21849 117939369 22201 2294019732 129 79 21849 117939369 22908 2294019732 137 33 Plates 1 7998 14489 1 8121 6 14486 9 10 15 16 1 MontageOffsets XYZScan End Region Info Fields Field Width 196 862745 Height 211 594005 Region Info Fields Field End Region Info Fields 100 5 RegionData6 0 08 01 2010 Class Name Class Colour Position X Fields 32768 8691 000000 15017 000000 124 830000 Fie
157. 777 17972 483843 21249 752833 17202 801290 21723 52833 17202 801290 21249 752833 17909 801290 21723 752833 17909 801290 22197 752833 17909 801290 8816 365106 20015 428112 9290 365106 20015 428112 8816 365106 20722 428112 9290 365106 20722 428112 9764 365106 20722 428112 11315 579162 19952 745559 11789 579162 19952 745559 11315 579162 20659 745559 Andor Technology Andor iQ USER GUIDE 913 Fields 32768 137 330000 Fields 32768 137 330000 Fields 32768 129 000000 Fields 32768 129 000000 Fields 32768 129 000000 Fields 32768 129 000000 Fields 32768 129 000000 Fields 32768 124 830000 Fields 32768 124 830000 Fields 32768 124 830000 Fields 32768 124 830000 Fields 32768 124 830000 Fields 32768 124 830000 Fields 32768 124 830000 Fields 32768 129 000000 Fields 32768 129 000000 Fields 32768 129 000000 Fields 32768 120 670000 11789 579162 20659 745559 12263 579162 20659 745559 18813 221330 19764 697899 19287 221330 19764 697899 18813 221330 20471 697899 19287 221330 20471 697899 19761 221330 20471 697899 21312 435386 19702 015346 21786 435386 19702 015346 21312 435386 20409 015346 21786 435386 20409 015346 22260 435386 20409 015346 8879 047660 22514 642168 9353 047660 22514 642168 8879 047660 23221 642168 9353 047660 23221 642168 9827 047660 23221 642168 11378 261716 22451 959615 Andor Techn
158. 8 bit image the highest possible threshold value is 255 For a 12 bit image the highest possible threshold value is 4095 After specifying the criteria for the merged image press Create to start the Merge The Create button will change to Stop press this at any time to abort the Merge The merged image will be displayed in the Image Window having pixel intensities equal to the pixel intensity of the first image plus the pixel intensity of each 411 Andor Technology Andor iQ USER GUIDE subsequent image at the corresponding coordinates The Preview button is used to display a preview of the merged image The Add button is used to add an additional image to the list for inclusion in the Merge The Delete button removes the selected image from the list The selected row will be deleted from the table The Save button opens a standard Windows Save File dialogue with which you can save the current Merge setup This can then be loaded for future use with the Load button On pressing Create the selected images are merged and the progress of the merge is displayed in the progress bar at the bottom of the dialogue 412 Andor Technology Andor iQ USER GUIDE 6 16 Photo bleach Compensation In time lapse imaging a fluorescence sample often live cells will commonly become less intense with time due to several factors including photo bleaching This can be reduced by minimizing the sample s exposure to the excitation
159. 9369 22908 2294019732 137 33 9 58 14 AM Position Y Position Z Andor Technology Andor iQ USER GUIDE 510 Fields 32768 124 830000 Fields 32768 124 830000 Fields 32768 95 670000 Fields 32768 95 670000 Fields 32768 95 670000 Fields 32768 95 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 120 670000 Fields 32768 104 000000 Fields 32768 116 500000 Fields 32768 120 670000 Fields 32768 119 320000 Fields 32768 87 340000 Fields 32768 116 500000 Fields 32768 116 500000 9639 000000 15724 000000 11190 214056 14954 317447 11664 214056 14954 317447 11190 214056 15661 317447 11664 214056 15661 317447 12138 214056 15661 317447 13689 428112 14891 634894 14163 428112 14891 634894 13689 428112 15598 634894 14163 428112 15598 634894 14637 428112 15598 634894 16188 642168 14828 952340 16662 642168 14828 952340 16188 642168 15535 852340 16662 642168 15535 852340 17136 642168 15535 852340 18687 856224 14766 269787 19161 856224 14766 269787 Andor Technology Andor iQ USER GUIDE 511 Fields 32768 116 500000 Fields 32768 116 500000 Fields 32768 116 500000 Fields 32768 112 340000 Fields 32768 112 340000 Fields 32768 112 340000 Fields 32768 112 340000 Fields 32768 112 340000 Fields 32768 133 170000
160. ATH environment variable The directory that needs to be added to PATH is C Program Files PI C 867 PI_GCS2_DLL where C 867 is the model code of the controller and may differ on your system lf you are unsure how to update the PATH variable in Windows please refer to your IT administrator or Andor Support 501 Andor Technology Andor iQ USER GUIDE Using the PI USB XY and Z devices in iQ To control the Physik Instrumente XY and Z controller in iQ Create or Edit a configuration using ConfigurationManager and include Physik Instrumente PXY USB Device and or Physik Instrumente PZ USB Device If you have both XY and Z control through a single PI controller unit please include both the PXY and PZ device options 2a Device Configuration Manager File Edit Settings Tools About Exit Create a NEW configuration lt lt Back Next gt gt Cancel Select the devices from the list that you wish to Use in this confi Available Auxilia Devices Nikon IntensiLight Device Nikon TE2Z000 Microscope Mikon TI Microscope Olympus Ki 6X61 Device Physik Instrumente Per USB Device mae hiysik Instrumente PZ USB Device Fiera Mewire When you launch iQ you will see device tabs Acquisition Auxiliary Devic called PI XY or PI Z To connect to the Po 2 e s 6 E 7 a a Current Live Snap Record Jt stay on lop the Device Select option to the controller you will see both the model and seria
161. Andor iQ USER GUIDE Tue ANDOR ia Andor 1Q2 USER GUIDE Last Update February 2012 1 Andor Technology Andor iQ USER GUIDE Oe OMB ic sate E EU IS AE AE AEAT E A ESE RAEE E IA EA A E E A RA AS 7 DE VVC OGUCHION dA AA a a a 8 L2 System Reguire MENIS bares sosa das aas aas Ad AA Aa TAM Ade dA e eaa A Teis ad dades 10 1 3 Access to Software and Installation cisisiscccsssccsccccctecsscassdscssccsedesscsscdevocsnededsacddecsseveotvcsessecesecsdadessesseeevousseeeass 12 LA HOW to USE this GU aaa a eiaa eaaa e e E 14 1 5 Andor ImageDisk Technology eescssssssececcccsssccceccososcccoccsosscececcssssecceccssssecceecosssscceososssseceosssssssecesesssssceeesssso 16 L6 Related Andor Systems Products sacisiscsccccsscccssessecscdecdevesasiacscccatascssbesecesuassescssscdecasteatenesseatannssassascseectecasteaeus 17 1 7 Sottware Development Kits SDK ua AA TA A E DD DA DEUDA RR 19 22 PHE USER INTERFACE iuri S tsetse seni NON aa afo A E E NE RA 20 2 1 User Management and Logon Procedure ssccsscccccsssssessscccscssssccssscccsccssscsssccccscsssscsssccccscsssccsssccssscsess 21 ZX SUAVE LO PEE EA T T EEEE E EE E E A E E T EEE E E 23 2 3 Concepts MANdOF 10 iaciissciecaseacaasessessasesbactasstssstasecusdeaseosessedesseqheseiesedausseopsusesseicddsssosteuateausiseceatessesseidsvacoacten 25 2 4 Supported Image File Formats ecocsssseeecccssssseceocsssssececosssssecoccossssececocssssecececsssssecocssssssecesossssseecessssssseesso 40 29 T
162. Andor iQ USER GUIDE 5 7 5 Analyzing Images Manage Analyse Review To perform a Spot Analysis using a protocol file selected previously in the Manage dialogue click on the Analyze tab the panel shown below is displayed e Analysis RGB Analysis Calibration Movie Editor Spot Analysis Manage Analyse Review Current Analysis Colona Analysis Results Number of images processed 0 Total number of spots 0 Number of spots in last analysis 0 Analyse Clear Data Select Images to Analyse CATEMPACOLONA AMI CATEMPACOLONAS PIC CATEMPACOLONA4 PIC CATEMPACOLONAS PIC CATEMPACOLONA2 PIC CATEMPACOLONAT PIC Number oflmages Selected 1 Individual images or a group of images are selected from the Image List as shown The images are analyzed using the threshold and feature filter settings defined in the current analysis protocol Data resulting from a previous analysis can be deleted from memory prior to performing an analysis by pressing the Clear Data button Once images have been selected for analysis the Spot Analysis is executed by pressing the Analyse button a progress bar continually monitors the status of the analysis Spot Analysis Analysing Images 1 of Displaying Image 286 Andor Technology Andor iQ USER GUIDE On completion of the analysis the Analyze panel dialogue is updated to show the number of images processed and the total
163. Andor iQ USER GUIDE A2 1 7 2 Software Protection Key Selecting Software Protection Key from the drop down list opens the Software protection Trouble shooter Page t Device Configuration Manager File Edit Settings Tools About Exit Software Protection Troubleshooter Read Your Software Protection Key iQ Camera Get Donale ID For Kinetic Customers who are Upgrading Existing Software Installing A Second Kinetic Product or g Activating Kinetic Plugins contact Kinetic with this Dongle ID number and you will be supplied with an initailisation code Initialise Dongle Select the Read button to list programs enabled on dongle Select Done when complete A2 1 8 Troubleshooting ActiveX Registration Device Configuration Manager File Edit Settings Bim About Exit Register Unregister Any Activex Component Configuration Register Unregister A Kinetic Plugin Device Nev File List Generator O eee ete Current Configurations The Tools facility is used for registering Andor ActiveX Components upgrading device drivers installing Andor Plug In software modules and troubleshooting 440 Andor Technology Andor iQ USER GUIDE hardware devices A2 1 8 1 Registering and Unregistering ActiveX Components t Device Configuration Manager Register Unregister ActiveX Components U IMPORTANT Ifyour device driver fails to register check that any Support Files and i nregister Done Drivers have
164. Andor iQ USER GUIDE Montage then every field will be updated in the Montage If it appears outside of the Repeat XY Action then it will only be called after the montage for all XY locations have been captured Return to Table Move Channel Move the devices associated with a Channel name into the recorded state This might include illumination and detection wavelength optical configuration camera settings etc Channels can also be assocaietd with FRAPPA actions or simply instrument states which are required for a given experimental protocol Return to Table Move Channel AND Snap Move the devices associated with a Channel name into the recorded state AND acquire a frame Acquring a frame with Snap executes Pre frame action e g open a shutter acquire a frame and Post frame action e g close a shutter Return to Table Move XY Move the XY stage to the coordinates specified in the Action parentheses The coordinates will be absolute if the Stage Registration wizard has been run In other words you will have a common zero position between sessions and the coordinates will be re usable By default the XY and Z coordinates at the current position will be selected and specified in the Action But you can drive the stage with joystick or GUI if one exists to a desired location and then right mouse click and select Record Current You can also select Move To from the right mouse Edit list if you want the stage to go to the recorded co
165. Box and can be assigned to a specific text label In the Protocol a Trigger IN command pauses the acquisition sequence UNTIL the Trigger IN matches the desired state or transition Hi or Lo or Hi Lo or Lo Hi A Trigger OUT command sends the appropriate signal level Hi or Lo or transition HI 35 Andor Technology Andor iQ USER GUIDE Lo or Lo Hi on the selected output s In this way synchronization events can be passed between systems or devices to ensure that image capture coincides with Specimen treatment or exposure whatever it may be The Trigger In Signals can also give rise to event markers which are stored within the acquired image sequence Event markers can be set manually by pressing the Space bar or may be associated with an external hardware input as discussed above All event markers are stored with a simple text description and can be edited and added to an image series retrospectively post acquisition Event markers generate graphical cues during Analysis through series or data review using the Navigator Further information concerning the use of Event Markers is provided in Section 5 6 2 3 6 ImageDisk lmageDisk is virtual memory system which uses a dedicated directory on the system hard disk or preferably on a separate fast hard disk or RAID array ImageDisk buffers images for rapid access during capture processing and analysis and provides permanent storage of data making it readily available between i
166. Brightest algorithm Alternatively Processing Algorithm C Darkest for moving cells then select none f None The brightest processing function creates an image which is maximum intensity projection through time in this case for each pixel The resultant image makes a suitable source for a mask and ensures that all regions which show activity at any time in the series will be ratio analyzed 388 Andor Technology Andor iQ USER GUIDE i Display Current Image Slice LTT TTT TTT Creating Running Brightest nDirn from CNTEMPSFURA TIF lf you choose the Brightest processing the mask source image will be created showing this progress dialog as it is processing The mask source image is converted to binary by a threshold operation as shown below The scroll bars are used to select the accepted intensity range for the binary image If the Brightest processing was performed the result will be a single mask at each wavelength shown left This is best for this example of stationary HEK cells If None is selected then the system produces a time series mask in which the image Threshold About Grey k 10 x at each time point is thresholded and a unique mask at each time point is used So the mask is a threshold with time making it suitable for masking moving cells Once you click OK in this dialog the mask wizard shows its final page for confirmation of the mask c
167. C Documents and Settings All Users Application Data Kinetic Imaging Configuration LUT 241 Andor Technology Andor iQ USER GUIDE 5 1 2 Image Contrast Control The Mapping and Automap options have the same functionality as the Mapping and Automap buttons described previously in Section 2 8 1 S2 Image Contrast Options View Invert E 1067 000 Min Exclude MA a ooa H Step G amma Coefficient I I 1 I 1 I 1 1 x 00 cot a oct Invert i W 1600 000 Min Eck SEs f00000 Step Gamma Coeticient ji ane a is ea 1 00 IV Smooth Use Region of Interest Map oh mouse up D selecting the Mapping menu item displays the dialogue illustrated above To invert the mapping flip contrast check the Invert option The scale of the mapping mode can exclude certain areas if the Exclude checkbox is enabled Selecting View gt Log Scale Histogram transforms the histogram Y axis to a logarithmic scale emphasizing the low frequencies Selecting View gt Zoom To Map scales the histogram X axis around the Min Max values as shown opposite From the drop down list below the Mapping Range scrollbars you can select the required mapping mode e g Gamma Square Square Root Sine Cosine Log e Log 10 Exp e Exp 10 and Linear The diagram below illustrates the various mapping modes that are available DA O Andor Technology Andor iQ USER GUIDE log 10 andlog e
168. Calculator Edit View Help value will need to be entered into Hex E Calculator Edit View Help the Devices ini 49304 O He OOct O Bin Degrees Radians Grads ow e O file as shown in step 6 below Before you can edit the Devices ini file as shown in step 6 below you have to create an iQ Configuration containing the Generic Shutter adapter Then Open iQ select the Configuration and continue to initialize all devices When you next Close iQ it will create the necessary entry in Devices ini file for you edit Then proceed to edit Devices ini Andor Technology Andor iQ USER GUIDE 6 Enter the value 48304 for the board above or your equivalent value from step 6 into the Devices ini file you will find this in the folder C Documents and Settings All Users Application Data Kinetic Imaging Common Open the file with Notepad find the entry shown below add the entry and then save the file Devices ini Notepad Seles File Edit Format View Help Interval 0 Genericshutter6 ShutterName Not Used HighState open LowState Closed ae POUL haa a E LowHi ghstate C losedopen Transitionbde lay 0 Interval 0 Genericshutter ShutterName Gr yvel select Highstate yel LowState cr HighLowst ate Gr yel 1 lad A EAEn TransitionDelay O Interval 0 Gener icshutter Selection 8 Line LPT Shutter Board 2 GenericsShutter Parameters File version 5 Acqui
169. Calibration create an FLZ Protocol Select 897_20X Protocol and insert Trigger actions Protocol Test Fast Trigger 2a Image Test Fast Trigger to control the start of a C Protocol Type Fast Time A Camera Binning Use Current A Wait Allocation On E Camera Selection Use Current capture Burst senda TTL In Pause Click OK to start protocol iiile pulse after each Burst and O Interval Oms fastest Repeat 10 times then wait for the next TTL Trigger In In0 High Device Setup A Trigger In In0 Low sequence to start the next Move Current Loop Protocol Channel Current Iv Display Image Burst cycle To do this Snap J Trigger Out Out0 HighLow create a New Protocol and End select the Fast Option as shown right Note we use the default Channel Current here so that we can adjust the camera settings without having to Record them in a separate channel But if you want to use a specific Channel then you can do so Acquisition Auxiliary Devices TO igi tt Ariel Curent n Live n Snap i iz Record New i 7 We can edit the Protocol to Set Repeat Camera Trigger DUS97_BY 4707 Settings Experiment 10 FLZ sequences or Bursts To Insert the eae Triggers and the required states simply use eet sua aes the Right mouse Insert at the required point Biwig in the Protocol sequence In the case x nZ above we wait for Input 0 to go Hi TTL 1 Open Cos
170. Channels Description 34 Repeat 170 umin 17 planes al Snap i End 2 di End Channel End End TO This is a regular Protocol for multi dimensional image acquisition By regular we mean that the frequency of acquisition is consistent for all fields Channels and Z values This kind of Protocol results in a single nDim because it is regular 214 Andor Technology Andor iQ USER GUIDE 4 6 9 Multi field Montage View and Multi Channel Py Protocol Test Case 11 MC MF Mon AF Mon View ma Image Test Case 11 MC MF Mon AF Mon View ie Frotocol Type Time Series ah Camera Binning Use Current Wait Allocation On Camera Selection Use Current Events fe sY Scan Test MF Mon oq Montage Acquisition Focus dP Pause Click OK to start protocol gi Repeat T 20 times O ms fastest J Repeat W Jo Repeat Montage Montage Positions 4 2 by 2 J A Positions 3 ia Repeat Channel Ch Ch Chi Channels Description al Snap i End Channel End Montage Montage View TED m oe a This is a regular Protocol for multi dimensional image acquisition Regular is as explained above It shows the way in which the XY Scan can produce a Repeat Montage loop when it is inserted into the Tree 4 6 10 Multi field Multi Channel Z Stacks with Autofocus Py Protocol Test Case 12 MC MP AF ma Image Test Case 12 T L MC MF AF Z i Protocol Type Time
171. DE The Settings section allows you to select the Control Port from the drop down list If Reconnect At Start is selected the device is automatically recognized when Andor iQ is opened If Report Manual Position is selected the stage coordinates will be displayed in the Communications section The Reset button can be used to revert back to the original settings Use the Load and Save buttons to load or save an image se Settings E l Control Part LOM Status Not Connected Reset The Settings Section also shows if Status Not Connected the device is Connected and its n Status Connected COM location or if the device is Not Connected The Reset button can be used to revert Reset back to the original settings Ooo O l Oe 477 Andor Technology Andor iQ USER GUIDE A3 6 3 The Ludl XY Stage Tab Ludi Shutter Protocol Action passinsa meei v Enter the Device Resolution microns step Ludi Shutter 2 Protocol Action Current for both X and Y The Default values are XvStage z WheelA Wheel B Settings shown in the picture below Device Resolution microns step 11 0000 Note The user must press Enter after Xx changing the X value and again after changing the Y value Verify that the Device Resolution microns step Y 10000 Values entered were set by checking the Establishing Communication Establishing Communication p Transmit Status Receive Ix Th
172. DG4 5 Settings The DG4 5 excitation source interface is shown left As indicated this controller handles both 4 and 5 filter versions of the device You select the 5 position version by checking the DG5 check box When checked a 5 filter disk will be shown The disks represent the filters within the device Acquisition Auxiliary Devices in the same way as the wheels above Setting up the disk properties Name C o O O So Value Units and Color is the same a m DGS described in section A3 6 3 above V Shutter Open Filter Wheel 469 Andor Technology Andor iQ USER GUIDE A3 5 The Prior Motorized XYZ Stage A3 5 1 Start Up of iQ At the start up of iQ the panel below is shown if one of the active auxiliary devices is a Prior motorised stage The user is requested to identify the home position of the stage This operation need only be carried out at the start of an iQ session or when the recognised stage position has x been lost and the device needs to Please reset your stage A be reset 1 Select a low magnification objective 2 Move the stage to the minimum Zero position and 3 Press OF Mote IF the stage is already calibrated you can enter Cancel The home position of the stage ae corresponds to an X Y Z position of 0 0 0 To zero the stage move the joystick accordingly In general driving the joystick to the top left corner of its travel and clicking on the OK button achiev
173. DYD CD ROM drives Performance Logs and Alert gy Human Interface Devices Device Manager Keyboards 5 Storage Mice and other pointing devices Removable Storage Monitors Disk Defragmenter B Network adapters Disk Management F Ports COM amp LPT Ta Services and Applications F Communications Port COM1 JY ECP Printer Port LPT1 F OXL6PCI9Sx PCI Parallel port yy MB Processors SCSI and RAID controllers indows XP LPT2 498 Andor Technology Andor iQ USER GUIDE To check the PCI card address select the card entry with left mouse click and then right click for a pop up menu and then choose Properties 3 499 0X16PCI95x PCI Parallel port Windows XP LPT2 Pr P In the Properties dialog check the fa General Port Settings Driver Details Resources I Resources tab where the IO hex OX16PCI95x PCI Parallel port Windows XP LPT 2 address Is listed as shown This Resource settings value will depend on the exact Resource type Setting A We eceo ece7 setup of your system lll O Range BCBS BCBF 1 0 Range BCEO BCFF J You can use Windows Calculator to convert the hex address to decimal which is the format iQ uses So enter the number BCBO here and enter it into the Calculator with the calculator format set to Hex as shown Then switch the Calculator to Decimal Dec as shown the result is 48304 in this case and this E
174. Description A n al yS IS gl Snap oop Protocol End Channel isplay Image j d j Booo Movie Editor Q End TO S pot Image Navigator Bi a ee E fos El Lo Es fes em fi ola es sz O Atoma 1000 355 20 119 184 e xe When you have logged onto Windows and iQ as described above the User Interface will open This consists of two windows which can be moved and resized as required If hardware is specified in the configuration then the Device Setup window will also be shown Options You can select which functions you want to be Functions Select the functions required visible on the iQ interface via the Function option in the Settings menu of the Control Window Please refer to Section 2 6 3 for further details The Movie Editor M Spot Analysis following dialogue is displayed enable the appropriate check box and then press the OK button Page tabs will be displayed for all selected OK Cancel functions 24 Andor Technology Andor iQ USER GUIDE 2 3 Concepts in Andor iQ Before we begin an overview of iQ functionality the reader should be aware of some key concepts These are straightforward especially if you have some experience of optical microscopy and once understood will clarify how to utilize iQ to best effect Please read this section The key concepts are Channels Scans Protocols Triggers and Events ImageDisk nDims Device Setup and ROI s and Classific
175. Ds iQ will automatically split files saved out from the lmageDisk into files up to the specified single file size limit There are practical limitations with too small a limit for example each single image from a camera could be over 1MB in size 544 Andor Technology Andor iQ How To Documents A5 12 Saving Multiple Images from the ImageDisk 1 Select ImageDisk from either the iQ Control Window or the Image Window Image Manager D KineticlmageDisk Shared File Edit Comments 2 The Image Manager TIA Type 3 8 bit grey pseudo Intensity 1 000000 320 window will appear 972401000000 um 3 Highlight a file you wish to save by clicking on its name 1 a Image List To highlight more than one Image Name Type Size mported mage 8 0 MB F MULTIPLE IMAGES GIMULATED PSF 20000 TIF ewa i H ACLEARVIEMASIMULATED PSF TIF file hold CTRL on the H COLOUR IMAGES MERGE DEMOBLUE TIFS H COLOUR IMAGES MERGE DEMOBLUE TIF i PSF_CROPS PROCESSED INC snap 1 11 Tse keyboard and click on PSF_CROPS PROCESSED INC snap 11 16 bit gr H COLOUR IMAGES MERGE DEMORED TIF2_21 8 bit gre 75 0KB H COLOUR IMAGES MERGE DEMORED TIF2_2 Sbit gre 75 0KB lt Close Show Rename Image Save Image Delete Selected Images contiguous files select the first file then hold SHIFT and click the last file in the block another file to highlight both files To select a block of 3 Click Save Image Save Image 4 The Multip
176. E lt Ea C Use separate image Mask Image Create Mask Image A Mask defines the region over which the processing will be executed It is a binary result of a pixel by pixel comparison against a constant value Mask where or it is a binary image or image series Whenever the result or image is 1 the calculation will be executed otherwise the pixel in the Destination image will be set to Zero This ensures that processing will be constrained to regions of the image where there is useful information and not background noise Select No Mask if a mask is not being used Select Mask where after background subtraction if you know the values you want to use Select Use separate image if you would like to create a mask with another image Use the select button to select another image from the image list Select the Create Mask Image to create a mask This will launch the Mask Wizard Section 6 1 2 376 Andor Technology Andor iQ USER GUIDE 6 10 4 Processing Summary Each option selected under the Source Background and Mask Tab will be previewed at the bottom of the Monadic Processing Window as shown below Source Background Mask Primary Background O SUMI Close Press the close button to exit the Plug In Help Press the Help button to access section reference for User Guide Process Once all selections have been made under each tab ds press the Process button A new window w
177. E2 for the first time you must Use the On Device Setup panel to initialize the COM port LED Intensity and GUI settings for wavelengths and colors LED 1 te as shown in Figure 2 LED 3 Figure 2 Shows the Andor iQ GUI for CoolLED PE2 control Pulse mode checkbox is circled in red Manual control is performed from the GUI shown while setup of the COM Port is done from the Settings tab Locked use lock button to access You can use either a standard shutter operation or a special feature of the PE2 unit 488 Andor Technology Andor iQ USER GUIDE called Pulse mode These modes are mutually exclusive and are recorded with the Channel See notes below regarding potential setup issues Standard Shutter mode set the Shutter to Open Close in your iQ Channel All commands and actions are coordinated by iQ via the USB interface This mode of operation and Pulse mode are mutually exclusive and if you have the Fire connected to the PE2 panel the system function will be compromised So make sure you have no cable connection to Fire e Note that when used with Luca R a 60 ms Shuiter transition delay setting will be required in the iQ PE2 controller Settings tab e Multi Channel Operation is identical to single channel in this respect Pulsed mode set the Shutter to Open in the iQ Channel and the Fire Pulse operation will gate the LED on and off e Make sure the Fire pulse cable is connected
178. ED 255 B G 0 while grey level 255 white is set to GREEN 255 R B 0 Use this Palette when setting up imaging conditions to detect when saturation occurs Red This is a monochrome palette using only the Red color component intensity 255 the maximum color brightness This provides an image shown in a manner that may be reminiscent of viewing a fluorescent image with the appropriate fluorochrome Green This is a monochrome palette using only the Green color component intensity 255 the maximum color brightness This provides an image shown in a manner that may be reminiscent of viewing a fluorescent image with the appropriate fluorochrome Blue This is a monochrome palette using only the Blue color component intensity 255 the maximum color brightness This provides an image shown in a manner that may be reminiscent of viewing a fluorescent image with the appropriate fluorochrome Bandit A color banded Palette the banded one in which groups of 16 grey levels are visualized as the same color This is useful for pseudo coloring and for visualizing and enhancing structural and intensity variation in images This is valuable for comparing changes to the image after a processing operation Furnace This Palette is a yellow brown table in which intensity is matched to brightness along the yellow brown color scale Furnace provides enhancement to the visibility of low contrast image structure lf none of the abov
179. ENE 123 Andor Technology Andor iQ USER GUIDE this file in the Dual Image Disk Plug In to perform alignment during the copy and merge steps to make the alignment process as close to automatic as possible for general use 11 Dual Image Disk Plug In applies alignment automatically Refer to Steps 5 6 above to E qual f Example explain the basic use of the Plug cate dual test_spli E fliprot 2ch ALI 22 E LastSession ali In E aes a E TestAligmentG51G52 ALI co E Files of type Alignment Files ali M Cancel KA 12 Select alignment file for isai zas ow Remote Image Disk 1 lt lt Copy C Remote Image Disk 2 automatic use To make sure O IS Alignment File Vv Apply Test ligmentG51G52 ALI m that images you merge from the Image List Updated Master and Slave system are pixel aligned as part of the copy and merge steps check the Alignment check box below the Constructor button To select the desired Alignment file click the square button in the Alignment control and use the file view and select the file named previously in step 10 13 Construct the aligned and merged files Now when you select the Master and Slave images which you wish to merge and click on Constructor button the copy will be done and the shift rotation specified by the file will be applied automatically 14 Remember to match the number of frames in n
180. Experiment Control FLZ Unchecked Experiment Control Trig Mode Free Run Experiment Control Ext Start Unchecked Use Frame averaging Unchecked Frames to be averaged 1 Fastest Grab Unchecked Available memory 1476 Tab Exposure End Tab Temperature Shutter Temperature Setting 20 Actual Temperature 20 Cooling Steady Fan Full TTL High Open Checked Shutter Open Transfer Time 60 Shutter Close Transfer Time 80 Tab Temperature Shutter End Tab Speed Amplifer Horizontal Readout 12 500 Vertical Readout time 0 889 ADC Channel 0 Vertical Clock Voltage Normal Output Amplifier EM Port Baseline Clamp Unchecked Tab Speed Amplifer End Tab Image Orientation Flip Vertical Rotation in Degrees None Tab Image Orientation End Image Info Window Left 0 Window Top 0 Window Right 1003 Window Bottom 1001 Andor Technology Andor iQ USER GUIDE Window Width 1004 Window Height 1002 Window BinningX 1 Window BinningY 1 Image Info End Image Bits 14 Bit Data TRUE Image Bits End First Frame SavedTime 1 8 2010 9 57 09 AM 15 First Frame End 2 This example contains the header of a 2 fields 3 by 3 montage image with 10 z steps per montage tile Name TL 3Ch MF26 08 01 2010 10 44 23 AM Type 14 bit grey pseudo Intensity 1 000000 lux x 1004 0 196078 um y 1002 0 211172 um Z 10 Montage 9 XY 2 Created Date 08 01 2010 Time 10 35 35 AM Created End XYZS
181. Fluorescence Recovery After Photo bleaching and Photo Activation In April 2010 Andor acquired Photonic Instruments and with it the patents and designs for two other innovative photo stimulation products namely Mosaic and MicroPoint 3 6 1 Mosaic solid state digital illumination Mosaic is a digital illumination system which utilizes digital mirror device DMD technology to control the illumination field of a fluorescence microscope Using laser or arc lamp sources Mosaic achieves real time and near diffraction limited resolution Mosaic can simultaneously and precisely excite multiple regions of interest with complex geometries parallel multi region illumination and allow simultaneous imaging Mosaic is unique yet flexible operating over wavelengths ranging from 365 to 800 nm with applications including uncaging photo conversion activation and bleaching Mosaic Is in use with laser scanning and spinning disk confocal systems as well as wide field or epi fluorescence microscopes iQ software drives Mosaic to map a mask defined in image view directly into the specimen plane at a rate exceeding 60 fos Its simple optical design is readily integrated with complex optical microscope systems designs to realize diffraction limited imaging with minimal loss over a broad spectral range Unlike traditional galvo scanning systems where pixels are addressed sequentially Mosaic provides truly parallel illumination of multiple complex ROIs MOSAIC expl
182. GUIDE The Interval Setup item enables a list of intervals to be created which can be subsequently accessed during the execution of a protocol allowing different repetition intervals to be used This function allows sampling rates to be adapted to the experimental condition Edit Intervals Repetition intervals may be inserted in the list by clicking on the Add Interval button and pressing OK Select interval length Interval lengths may be specified in milliseconds e seconds minutes or hours Cancel Intervals are removed from the list by highlighting the unwanted item and clicking the Delete Interval button During a live acquisition sequence the list of repetition intervals is accessed by right clicking on the Interval item in the Protocol Tree 52 Andor Technology Andor iQ USER GUIDE Po Protocol Test The appropriate sampling interval is mees Upe Tene Seis selected by clicking the right mouse ty ab Camera Binning Use Current Wait Allocation O ae Camera Selection Use Current E EO Events i e AY Scan MPs3F Mon EE Montage Acquisition age Focus i sean is to Se ee button a EH Repeat Montage o beg es Montage Positions 4 2 by 2 J Fie er Positions 3 a 3 Repeat 9 unin 11 planes E a Move Channel Current 2 Snap ead a End 7 Bese a End Montage oe a End Y SS a End TO 2 5 4 The Wizards Menu The Wizards menu offers access to all calibration
183. GUIDE 6 14 Plug In Registration and Access The Plug Ins are accessed from the Andor iQ Plug Ins menu as shown below Options RGB Merge Photobleaching Compensation Plug In modules provided with Andor iQ are automatically registered during start up of the program and are ready for use The RGB Merge and Photobleaching Compensation functions are not dynamically loaded and appear automatically on the Plug Ins menu each time Andor iQ is executed Modules currently under development will be available for purchase in the future and can be registered using the Configuration Manager The use of the Configuration Manager is discussed in Appendix 2 Instructions regarding the installation of any subsequent Plug In modules will be provided at the time of purchase Analysis Plug Ins Help Selecting the Plug Ins menu item allows any of the Pens RGB Merge activated functions to be selected from the list l l Photobleaching Compensation 6 14 1 Options a 4 Mask Wizard Clicking on the Options menu item e vi Dimension Editor enables the appropriate Plug In function to be activated by enabling M Clearview Deconvolution selector l the associated check box as shown W MiewiOrthogonal Slice View ha in the example 409 Andor Technology Andor iQ USER GUIDE 6 15 RGB Merge This is used to merge multiple images and or multi dimensions e g Z T Field selected from the Image Disk The merged image will be disp
184. ICE SETUP 2 2 0 c0scccnssessecccnsnesescecnnesseccennnsseeccennnassecesaeneseas 443 ASA Hardware DEVICES asico ouien aeaniee aana aaa iaeiaiai oaa oaaae ar o daaa aaae 444 ASZ ANdOr UX OW s a a a a 446 A3 3 DIO and DAC Card Installation for PCU and FLZ sssesssesessssssssssssoocccscsssssssssssssssoscssesssssssssssssssssssee 459 AS4 TheSu tter Filter DEvCeS as a EU a a a A NO Oa IDEN 464 A3 5 The Prior Motorized XYZ Stage sssssccccccsssecccccccsssccccccsssssceccocssssceccccssseseececssssececeossssececcocssssseeccossssecee 470 A3 6 Ludl XYZ Stages and Filter Wheel ooooossssssssecececcccsocsssssessecccecocosssosssssssscccececsossssssssssssseccecosssssssssoe 474 ANIT AST ACY Zi SUACES Arena AA E TE scbeeasansecusseeneeeds 482 AS Thor Labs LEDDI controll r sissssccscsccnccisctisccavessscdescssvedesdebvessssessiessscvevecsvesevessveusvacdssesvecseaceveccdasaveastasssess 485 A3 9 CooLED PE2 and PE100 LED illuminators sssssssosseeessssssssssssooscscccsscssssesssssssosssecsssssssssssssssssssse 488 4 Andor Technology Andor iQ USER GUIDE A3 10 Andor Differential Spinning Disk DSD ccssssssscccsssssssssccccccccsssssssccccsccssssssscccssscsssssssscssseeees 491 A3 11 Metal Halide Source Prior LumenPro ccccccsscccsscccsscccscsccssscccsccccscscccscccceccccssccccccccescccscccesescess 494 A3 12 Andor PZT X00 Piezo Stage CONC ON sass dccccccnse scsssccescccsccsuscestceescesessucassescan
185. Next to continue 2 Series options Zz Select Start 331 6 Under the Z Series options Select the Start and a aS End point for the Z scan Also choose one of the Number of Planes saen E agi Stack Definition parameters AZ Number of planes ae Stack Definition select parameter to be computed or End Position ae C Number of Planes File Image List Settings OC Wizards Plug Ins Help EIROA Setup Wizard Scans Scan Manager aE s Press Next to continue New Edit Delete Close Scan Summaries Current Scan New Scan is then added to the list under Scan powerpoint aa S Manager and can be selected in setting up E A CON powerpoint an ais amp Scan test6 posn Dip Getz Goer protocols amp Scan zredonion 534 Andor Technology Andor iQ How To Documents A5 4 How to Set Up a Prototype Protocol Andor iQ 1 10 0 Andor Came 10 x 1 Select Protocols from the Wizards Fie Image List settings QC Wizards Process Analysis Plug Ins Help Setup Wizard menu or from the Acquisition tab click the Protocols Protocol Manager Protocol button to open the Protocol Manager Delete Copy Select Cancel 2 Select New to create a new Protocol Protocol Summaries j Current Protocol FastTime 4 1 4 Protocol 110 Mivv Mon UD1 NoZDC H P Protocol 1 New Protocol 4 Protocol f Protocol 1 Select name for new Protocol 4 Protocol 1 Fast Protocol f Protocol
186. Note this can be a remote or network drive ee select the drive and folder then press OK The folder name will be displayed in the Auto Save text box in the Acquisition dialogue Auto Save J IEA Inetic WOD ata 58 Andor Technology Andor iQ USER GUIDE 2 6 1 4 Protocol The Protocol section of the dialogue is used to set up and control experiments Protocol Pressing the Protocol button will launch the Protocol Manager Es Protocol The Protocol Manager allows you to Select or Delete a Protocol Q Run from the list or Create a New Protocol New Protocols are e Channel cod ey Sean created as Prototypes which are then edited in the Protocol Tree by Edit Insert Delete and other keyword specific actions After hat eines cone completing the setup you will return to the Acquisition dialogue where the Experimental commands are displayed in a tree type Loop Protocol M Display Image structure Further details regarding the use of the Protocol Eo Manager are described in Section 4 4 Note that any Channels or Scans to be included in the Experiment must be set up first from the Channel Section 4 2 and Scan Section 4 3 Managers press the Channel or Scan buttons respectively to E Tr Save As a Oriens wi Uptions access these functions 2 6 1 5 Run Channel Scan Device Setup Store Store As After an Experiment is set up press Run to start The button label will change to Stop To
187. Pint Preview Printer AWEB SERVER SBrother HL 1 Setup Print Onentations i Portrait tf Landscape Bie ES Close Beset Maime M View Margins W Proportional pa ee ee Andor Technology 302 Andor iQ USER GUIDE The Save Plot Data and Print Plot functions are also accessible from the menu bar Scatterplot of Area and Integrated Intensity File Selection Save Plot Data Print Plot eee ee ee ee ee 3 800 a 5 7 6 5 Show Images The Show Images function enables features to be visualized together with their bounding rectangles and cell numbers assigned previously during quantification This function is particularly useful for the analysis of temporal data where a significant change in spot characteristics may occur at a particular point in the time series Clicking on the Show Images button opens an Image Review window which enables the selection of the required data set A window following the analysis of an individual image is shown below T a The appropriate image is selected by clicking Select Processed Image to Review CATEMP COLONA1 PIC_Colonal on the file name with the left mouse button Clicking on the View button results in the display of the selected image in the Image Window An example is shown over 303 Andor Technology Andor iQ USER GUIDE Kinetic Imaging Spot Beta 1 0 File Image List Region Colours Tools Iss lmlele e ___ttsor
188. Press PREVIOUS FIELD to select position and mowe to Using the joystick move the XY stage to previous field the position of the first field The x and y Please ensure image is focussed as z position will Peed HE ENEIEHIE positions will update in the blue x position 52269 highlighted text boxes as will the z tion 27021 ee PESA Current Field 1 position if you have a Z motor z position 0 Next Field gt gt Z Position lt lt lt lt lt 0 gt gt gt gt gt gt 14 600 0 1 p 3 During the field definition iQ will show a 15 000 assas kenara paasa Jessie graphical view of the current stage 15 200 4 zu 15400 position and the fields selected so far 15 800 Liota adaa l as Shown left in this case 4 fields have been defined and you can see their 13 000 13500 14000 14500 15000 15500 16 000 relative positions in stage coordinates j Copy to Clipboard Save Chie 1 000 um 1mm This graphic will update whenever you run or change the scan in a Protocol Note When editing a multi field scan iQ will allow you to choose to reposition the stage at the previous XY fields listed in the scan This is essential if you are adding fields to a scan You can also choose whether to allow iQ to re use previous focus or Z positions during repositioning We make this an option to reduce the risk of resetting incorrect focus positions which may damage the objectives
189. Q USER GUIDE File Image List Settings QC Wizards Plug Ins Help Setup Wizard Multiell Petri setup well settings lt lt Back Next gt gt Cancel Well Pattern summary of well settings Name Costar SSF 3x2 35 29 settings Type Multi ell Humber of ells Mx 3 My 2 Well separation 0x 39 mm Dy 39 mm Well shape Circular diameter 35 A summary of the well settings is now displayed Press Next to continue Once the Well or plate pattern has been defined or selected if you are re using or editing the pattern then the user needs to show the system the top and left edges of the first and second wells of the first row of the pattern This is needed to allow iQ to lt lt Back Next gt gt Cancel Registration point offsets The registration point offset is the distance from the stage registration point to the top left corner of the first well 1 Steer the stage so that the top edge of the first well touches the horizontal line 2 Click Record Y Offset Y Offset 0 mm 3 Steer the stage so thatthe left edge ofthe first well touches the vertical line 4 Click Record X Offset x Offset 0 mm deal with pattern Skew i e rotational offset relative to the camera and stage coordinates This can occur in various scenarios when a pattern is fixed manually to a carrier or substrate and is not perfectly aligned There are two dialogs to handle thi
190. Q which one to use by selecting the device in the radio group then highlighting the required calibration in the tree and clicking the Select button 3 6 8 2 Multiple Device Calibration Dependency Note that a device calibration may be dependent on certain other system variables and calibration may be lost if these variables change For example if a FRAPPA device has been installed in the optical patch between sample and camera and a calibration is carried out for a rear mounted FRAPPA device e g the Mosaic or MicroPoint then the calibration may be lost if a different FRAPPA calibration is selected which changes the FRAPPA registration position 147 Andor Technology Andor iQ USER GUIDE 3 6 8 3 Including Multiple FRAPPA devices in a protocol Multiple devices in a protocol are distinguished from one another via a device keyword When the protocol switches to a FRAPPA channel the current device will switch to that indicated by the keyword and the selected calibration for that device will be used The device keyword is set in the Channel Select dialogue which displays when a FRAPPA is inserted into the tree Channel Select iy Protocol Protocol select channel oa Image Protocol FRAPPA Region fF Protocol Type Time Series Selected All wf Camera Binning Use Current Wait Allocation Off Camera Selection Use Current FRAPPA Device toga Events COTOS EEES dP Pause Click OK to start protocol 9
191. Repeat T 1 time O me fastest ae dr Frappa rept Mosaic al sical pis Ao Move Channel Current EE al Snap o Cancel End T The second keyword All in the above example indicates whether to FRAPPA the currently selected region or all of the defined regions 3 6 8 4 Interactive or Click and Fire FRAPPA with Multiple Devices When a FRAPPA region is selected on the ImageView the user can perform manual FRAPPAs via the right click context menu If the user has multiple FRAPPA devices in the system he will need to set the current device He can do this either in the Select FRAPPA Device dialogue as selected from the Wizards Menu or more directly using the right click context menu as shown below 148 Andor Technology Andor iQ USER GUIDE 2 0 333 x 512 0 333 2D Region View Fast MIP a Niele Ooo T i m Rol C Select Tool Auto Map Copy Edit Delete Delete All Classify Load Save Set Reference Frappa This v Frappa On Mouse Up Frappa Device v FRAPPA Frappa Channel MicroPoint Frappa All Regions 149 Andor Technology Andor iQ USER GUIDE Ee ANDOR ee Ie CHNOLOGY 4 Acquisition Tools 150 Andor Technology Andor iQ USER GUIDE 4 1 Configurations When you create a Configuration in iQ 2 you choose all of the hardware components you want to control An example is shown in Figure 4 1 1 You can create multiple config
192. Scatterplot of Area and Integrated Intensity File 300 Andor Technology Andor iQ USER GUIDE ae nr ne enna ld To define an area simply press Select Area so that the cursor changes in appearance then using the left mouse button click once in the plot area the next click will draw a line between these two points and so on To close the area press the Right mouse button 100 000 200 000 300 000 400 000 500 000 Integrated Intensity Deselect Area Spots 102 OK Cancel Save Plot Data Print Plot The Deselect Area button will remove the areas from the scatter plot The Select Area and Deselect Area functions are also accessible from the menu bar Scatterplot of Area and Integrated Intensity File BET Data Deselect Area To save the Scatter Plot data press the Save Plot Data button A standard Windows Save File dialogue is opened which you can use to save the data in the usual manner 301 O Andor Technology Andor iQ USER GUIDE Save Scatterplot Data Save jr A Kinetic a colona sip Save as type Spots E xcel scatherplot data xlp Cancel All the measured parameters specified previously in the Review panel corresponding to each data point in the scatter plot are saved to the data file The Print Plot button enables the scatter plot to be printed for further inspection and comparison An example Print Plot window is illustrated below TeeChart
193. Select Z Device Device Setup lt lt Back Next gt gt Cancel Montage Focus Definition Please steer the stage and focus on at least 3 points to allow a focus plane to be calculated Press NEXT FIELD to select position and promptto next reference point Press PREVIOUS FIELD to ignore this position and move to previous reference point x position 15198 234 y position 2081 9 964 z position fi 08 33 Next Field gt gt mZ Position TAAR E SS 108 33 F IDY use the field values you specified In both Edge and Region cases you can give iQ some help with focus control and it will fit a best approximation focus plane to try to keep the feature in focus throughout the scan To make use of this feature use the joystick to move to some fields within your scan and focus the feature of interest and click on Next Field You will need to specify focus for at least 3 positions to allow fitting of a focus plane lf you want to ignore the focus plane tool then simply Click on Next at the top of the wizard panel Then iQ will You can use the microscope hardware Autofocus to compensate for drift and in that case iQ should apply the focus plane taking into account the drift corrected Z values In all cases you can add a Z scan in Time Lapse or utilize FLZ to ensure that you have sufficient Z range to obtain a focus image across the entire feature 179 Andor Techno
194. Settings dialogue Pixels with an intensity outside the limits are ignored Maximum Threshold the highest pixel intensity threshold limit specified in the Settings dialogue Pixels with an intensity outside the limits are ignored Region defining coordinates the X and Y coordinates of the top left bottom and right points of the region 260 Andor Technology Andor iQ USER GUIDE 5 5 Analysis Options 5 5 1 The Options Page Analysis Tab The Options page is where the Analysis options are specified and the statistical values to be calculated are selected All options are selected and deselected by a mouse click Selected options have their checkboxes labeled with a tick Graph Data Options iw Automatic Directory Selection Group Analysis Greps Per Second iw Background Correction Threshold iw Clipping Mean Intensity Peak Intensity SD Area ZATEA Integrated Intensity lntegrated Intensity Integrated Intensity Per Unit Area Return To Graph Channel Selection Cho 400 rm Solid ie Ch 325 nm Dash Automatic Directory Selection Once an analysis has been performed pressing Save opens a save dialogue window allowing the user to specify a directory and file name in which to save the data If Automatic Directory Selection is selected the default path to which the data will be saved is C AndorBio imaging iQData 261 Andor Technology Andor iQ USER GUIDE Please note any
195. Shutter Temperature Setting 20 Actual Temperature 20 Cooling Steady Fan Full TTL High Open Checked Shutter Open Transfer Time 60 Shutter Close Transfer Time 80 Tab Temperature Shutter End Tab Speed Amplifer Horizontal Readout 12 500 Vertical Readout time 0 889 ADC Channel 0 Vertical Clock Voltage Normal Output Amplifier EM Port Baseline Clamp Unchecked Tab Speed Amplifer End Tab Image Orientation Flip Vertical 527 Andor Technology Andor iQ USER GUIDE Rotation in Degrees None Tab Image Orientation End Image Info Window Left 0 Window Top 0 Window Right 1003 Window Bottom 1001 Window Width 1004 Window Height 1002 Window BinningX 1 Window BinningY 1 Image Info End Image Bits 14 Bit Data TRUE Image Bits End First Frame SavedTime 1 8 2010 10 35 36 AM 31 First Frame End Montage Adjusted true Montage End 528 Andor Technology 929 Andor iQ How To Documents ee ANDOR 2 TECHNOLOGY Appendix 5 How To Documents Andor Technology Andor iQ How To Documents A5 1 How to Set Up a Channel 1 Select Channels from the Wizards menu Pee eee Setup Wizard or from the Acquisition tab click the Channels Channel Manager Channel button to open the Channel Manager New Edit Copy Delete Close Channel Summaries Current Channel 2 Select New to create a new Channel aiis Channel FITC 3 Enter a
196. TFIELDBRIGHTFIELD AFER TIF_Fi CAIMAGES DYO V1 49 BRIGHTFIELDBRIGHTFIELD AFER TIF CAIMAGES DVD V1 4S4FILTERSSOURE RUNT_ 250M TIF CAIMAGES DYO V1 4S FILTERS420_GELIPG CAIMAGES DYO V1 4S BRIGHTFIELDSBRIGHTFIELD AFER TIF_Fi CAIMAGES DVD V1 4S BRIGHTFIELDSBRIGHTFIELD AFER TIF Disk Selection Show Refresh Refresh Local Image Disk O Remote Image Disk 1 lt lt Copy Hame Construct Constructor Alignment File W Apply Test By Remote Image Disk 2 Image List Updated Just like the ImageDisk itself the location of any other Image Disk s must be configured in the Global Configuration Settings using the Configuration Manager If two PCs are used then they must be connected via a Microsoft Network and the Master PC will have the Slave ImageDisk disk drive Mapped in Windows Explorer and hence assigned a drive letter e g Z or K See the chapter 2 for more 407 Andor Technology Andor iQ USER GUIDE details about the setup of the dual ImageDisk If both instances of iQ are on the same PC they will acquire to the Local ImageDisk as shown here Once opened the Dual Image Disk plugin can be seen as above On the left are the contents of the local Image Disk Clicking an image in the list and pressing Show will load the selected image into iQ s Image Window Pressing Refresh will update the list On the right it is possible to see one other Image Disk Selecting the appropria
197. To Exposure 00 Boo oo os Z Control Eo Start O EEN EE M Move 1st Step 25 suo Set As Computed Param Planns i a Nyni Fam Ea Nyquist Formula for Step End 100 F Step through planes O Go Centre lam didi O Time Burst 0 053 s Save to file Load from file 10 Note that the Sequence tab above shows only a single wavelength L for capture so this is a single acquisition with excitation exposure and emission settings as indicated on the interface If Exposure is not set on Sequence tab then it uses the value set in the Current channel 20 ms on the previous page 11 Now everything is setup to run a series of 10 Bursts with 100 frames per Burst i e 1000 frames will be acquired but the system will require Trigger Input 0 to show a Lo followed by a Hi TTL level before each Burst At the end of each Burst iQ will issue a 10 ms pulse indicating to the external hardware that it should initiate the next Burst when ready 12 On the last page over the iQ Control Window shows the Protocol being executed and at a synchronization step after the first Burst has been captured The Protocol Status shows iQ is waiting for the Trigger Input 0 High signal to start the next synchronized acquisition Burst It also shows that iQ has captured 100 frames or one Burst and the elapsed time is 6 172 seconds since the start of the Protocol 557 Andor Technology Andor iQ How To Documents 13 Hopefully it will be Andor 1
198. XP File KI_MERGE File KI_NODE File KI_RATIO File CHARTOPTIONS File KI_EXP File KI_MERGE File KI_NODE File KI_RATIO File LST File Configuration Settings CHARTOPTIONS File KI_EXP File KI_MERGE File KI_NODE File Date Modified 8 5 2005 3 21 PM 8 5 2005 3 21 PM 8 5 2005 3 21 PM 8 5 2005 3 21 PM 8 5 2005 3 21 PM 8 5 2005 2 48 PM 8 5 2005 2 48 PM 8 5 2005 2 48 PM 8 5 2005 2 48 PM 8 5 2005 2 48 PM 8 5 2005 2 43 PM 8 5 2005 2 43 PM 8 5 2005 2 43 PM 8 5 2005 2 43 PM 8 5 2005 2 43 PM 9 9 2005 11 56 4M 9 9 2005 11 56 AM 9 9 2005 11 56 AM 9 9 2005 11 56 4M 9 9 2005 11 56 4M 9 8 2005 5 45 PM 9 9 2005 7 28 PM 9 9 2005 12 15 PM 9 9 2005 12 15 PM 9 9 2005 12 15 PM 9 9 2005 12 15 PM E E Start Menu E FastS0 KI_NODE gt Templates lt v 2 Protocols and Channels are stored in Hidden Folders which can be revealed in Explorer under the Menubar Tools Folder Options View provided you have permission for this action on the PC The folder which stores the Protocols is named C Documents and Settings All Users Application Data Kinetic Imaging iQ Configuration where Configuration is the chosen name of the configuration as shown in the figure below This name does not have to be the same between machines or users but MUST have exactly the same devices selected 3 Once you have identified the correct folder s you will notice that the folder s contain several groups of files with the same name
199. a region of the specimen which has been bleached A bleached region will appear darker than the rest of the specimen and should be localized Note if the bleach cannot be seen you may need to scroll the XY stage a little or increase the FRAPPA time if the bleach is still not visible This step of finding the initial bleach may be tricky the first time you calibrate the system Once the bleach spot has been identified in the field of view then you must create corresponding pixel coordinates and angles for the calibration procedure To do this for each bleach point region click on your best estimate of the region center with the marker and then click on the button labeled Add Calibration Point Select 0 1 degree steps and incrementally adjust the two axis controls Using 0 1 degree steps establish which directions correspond to the camera X and Y 139 Andor Technology Andor iQ USER GUIDE directions Each time you increment the angle by one or more 0 1 degree steps then FRAPPA locate the bleached region center with the and click on Add Calibration Point Create at least 4 and preferably 7 8 points for calibration Use the angle adjustments to create bleach points at the corners of the image as well as around the center When you are satisfied that you have sufficient points over the image then click on Next and the FRAPPA Calibration will be created by the system and used henceforth whenever a FRAPPA act
200. a montage scan you MUST successfully run the Stage Alignment Wizard In the single scan case as illustrated in the dialog on the left you have a choice of three ways of defining the Montage scan fields 4 3 5 1 Linear Montage Scan File Image List Settings OC Wizards Process Analysis Plug Ins Help Setup Wizard The Linear method is designed to Set up XYZ options gt support observation of filamentous structures vessels or regions of SelectZ Device Device Setup Z 3 feature boundaries We show some Next gt gt Cancel Montage Definition examples of vessels from H E stained brain sections later in this iQ will automatically adjust to the epeseavaiane chapter Other users are exploiting Luca 40X Linear Specify the start and end of a linear feature this technique for applications I Sts ate aca al including imaging of combed DNA C Region Specify the width and height of the region Use the stage to define the boundary or centre of the region Select Linear and then click Next Field Overlap 5 176 Andor Technology Andor iQ USER GUIDE Now move the stage with the joystick and setup XYZ options choose start and end fields defining the Select Z Device Device Setup line to be scanned You should ensure lt Back Nee that you focus each field iQ will Montage Linear Definition inte rpolate the Z values for the intermedi
201. aRaS 541 A5 9 How to Import Multi dimensional Images Part 1 oosssssececccssssccecccsssssecoccsssssceecossssseceocsssssseeeossssssee 542 A5 10 How to Import Multi dimensional Images Part 2 ssssseccccsssssccecccsssscecoccssssscceoosssssccecssssssseecossssssee 543 AS 11 How to Adjust the File Size Limit cccisscccccissasccscceascvedesancvccesasssccesascascvsacecesesescaccscececsdeasecvessessevedssassescess 544 A5 12 Saving Multiple Images from the ImageDIsk scccccssssssssssccccssssssssssccccscsssscsccccsscesssssssccsssseees 545 AS 13 How to Save JPEG or BMP Images sssicccscssccuiscsascedecsccsncessassncsccascscscsssceddusdecncssassensduadectsscesssedsesassaceeas 547 A5 14 How to Copy Images to the Clipboard cccccccsssssssssccccsssssssssccccsccsssscssccccsscssssscccssssssssssssccsseseees 549 A5 15 How to Share and Backup Protocolls ssccccccccsssssssssssccsssssssssssccccccssscssssccccccssscssscccsccssscsssscocssssess 550 A5 16 Setting FLZ Frame Transfer FT Blanking cccccccsssssssscccccscsssssssscccccccssssssccccssscsssscssccsssseees 552 A5 17 How to Configure Triggered Looping with FLZ ssssssscccsssssssssssccccccsssssccccccsccsssssssscossesees 554 A5 18 How to Debug DSD Calibration Andor internal sscccccscssssssssssscccccccssssccsccccccsssssscsccsssssees 559 A5 19 How to Setup MicroPoint for Uncaginng ccccccsssssscsscccssssssssccccc
202. ack Next gt gt Cancel Set up Thresholds Bright Objects Dark Objects Lower Threshold 3150 8 000 7 500 aor ace 7 000 6 500 6 000 5 500 5 000 4 500 1 500 1 000 4 m e e e e dj e oe oe oe oe oe oe je oe oe e e e O e o e a a a ea oe Aa a oreo Bee a a a Sse 00 a DOS E ES R Seuss eqt een eee fen eeneqeeceene sf Upper Threshold fanon A RAE SA See ae OS T FSR Sees Peer Bao eee 3 500 4 1 4 3 000 4 4 t 4 2 500 f lt 0 4 2 60a A EA fan 2 000 4 4 fonnnen denen ne nbe 1 000 1 500 2000 2500 3 000 3 500 4 000 An image histogram is displayed lower and upper threshold limits represented by two vertical lines The green and red lines representing the Lower Threshold and Upper Threshold respectively The threshold limits can be adjusted using the spin buttons or alternatively typing new values into the respective text boxes the specified threshold values is displayed to A binary image created using the right of the image histogram panel As the threshold values are adjusted the image is updated This can be compared to the original image and the effects of the various thresholds optimized A grey level image and a complimentary binary image are shown below 280 Andor Technology Andor iQ USER GUIDE Grey level image Spot Analysi
203. age Oy B 38574411 Move TO P i Record Current on e 7 4952211 i oo O eY B 27625 5040 0 oe fell 3 Store As oe wels Sie ps EA Wells Highlighting a coordinate set and Be Welg e a selecting the Move To option moves i oi rd the stage to the coordinate positions listed 4 3 7 2 Modifying a Multi Field Scan An expanded Protocol Tree corresponding to a Multi field scan experiment is illustrated below The XYZ coordinates for each location in the six position scan are displayed in the tree Selecting the coordinate of interest and clicking the right mouse button accesses the Move To and Record Current commands as described previously in Section 4 3 7 1 186 Andor Technology Andor iQ USER GUIDE Protoca Protocol N Aun Channel Scan Device Setup Pe Protocal protocol A Image image ane ff Protocol Type Scan ff Scan Multi Field n Pause Click OK to start protocol 04 Interval 264 ms fastest l Repeat 7 times apes Repeat 7 gt DS Positions 6 Xr 2 82924434 Move Te D sty 3 B2924 482 Record Current of Xv 4 75651 48237 1 6 ow JB sev 5 75651 50676 1 6 JP SY 6 80594 47129 1 E a po Channel Ca380nm Protocal M Display Image Store Store As 4 3 7 3 Modifying a Z scan Protocal protocol Protaco A Image image ame elf Protocol Type Scan AF Scan Dave Z series Proto
204. age Release the button to complete The selected stamp will appear in the drawn box Image Mame Creation Date Right clicking on the image or a selected ROI will also bring up SEE the same menus as discussed in Section 2 10 1 To editthe stamp select edit and the user can select Time Exposure Event Event Current Date Current Time 99 Andor Technology Andor iQ USER GUIDE 2 10 15 The Define Scale Bar Button T m scale bars can be drawn on the image with measured length readout in Euclidean distance point to point 2 10 16 The Define Scale Line Button s p Scale lines can be drawn on the image with measured length T im o g readout in Euclidean distance point to point as shown below BI MUA Riel DIOS TLi lamje 2 10 17 The Current ROI Button e Paula drosophila PBC VerticalFlip Rotate180 16 Bit 642 0 107 x 1020 0 107 4D File Edit ImageList Region View Fast MIP Bw WA Nel DOOS T i ma O po i Background _Crop _Cycle _FRAPPA Feature 1 Edit Classifications A mouse click on the Current ROI button enables the current ROI displayed in the image to be reassigned to any class in the definition list Alternatively the current ROI can be reassigned to a new class The creation and classification of ROIs was described previously in Section 2 8 4 100 Andor Technology Andor iQ USER GUIDE ba IECGCGH HNOLOG Y 3 Calibration and Alignment Tools
205. age is focussed as z position will be recorded if available x position 1847 3 y position 21 37 85 z position fo Current Field 1 us Field Next Field gt gt 7 Set up Multi Field Options by moving stage to desired location and selecting Next Field Continue until all fields have been selected 932 Andor Technology Andor iQ How To Documents Next Field will change to Finish for the last Field Setting Current Field 4 lt lt Previous Field Finish 8 New Scan is then added to the list under Scan Manager and can be selected in Bere Scan 4 well slide HS Scan 5 field scan 5 46 Scan Carrie s field scan x User Defined Fields 4 Setup Channel bright field Scan multi well slide Scan Test 01 setting up protocols 533 Andor Technology Andor iQ How To Documents A5 3 How to Set Up a Z Series Scan 1 Select Scan from the Acquisition Tab or File Image List Settings QC Wizards Plug Ins Help Setup wizard the File Menu to open the Scan Manager Scan Set Up New Scan 2 Select New to create a new scan AEN Scan Setup Please enter a name for the new Scan i gt powerpoint 3 Select Z Series Scan option Scan Options M Multi vvell f Petri Scan M Multitield Scan 4 Enter a name for the new scan iv Z series Press Next to continue zZ 5 Select Channel to go live with for Scan setup iQ will use the Last Channel in use Press
206. ain differences between these codecs Size relative to uncompressed file Quality Intel Indeo Approx 1 14 AVI Movie Save The results will vary depending on the sequence Saving AM led i aving you wish to export Extremely detailed images with larger uniform areas will Compress better than highly detailed images with small well defined areas It would be beneficial to experiment with a short frame sequence using different codecs and different quality settings in order to achieve the best size quality ratio A suggested starting point would be the Cinepak codec with a quality of 75 Following the specification of a frame rate and compression format a progress bar monitors the AVI Movie Save process 273 Andor Technology Andor iQ USER GUIDE 5 The Spot Function The Andor Technology Spot software was originally developed to quantify ploidy chromosome number distribution in cell populations especially tumor cells The basis of this approach has been proposed in the analytical cytometry literature for some time By staining the cell nuclear DNA with a fluorescent probe and viewing in a fluorescence microscope it is possible with image analysis to quantify the fluorescence intensity This is after all the basis for flow cytometry and fluorescence activated cell sorting FACS However using a microscope image cytometry approach it appears that analyzing only a few hundred cells could perform a comparab
207. aled into the image A Window as shown below 94 O Andor Technology Andor iQ USER GUIDE BK NAW Nile DoS TI ilm Ro L O ry W Multi i lt bal LEC ag 2 10 5 The Define Point Button ko F gt 2 The Define Point button allows you to define specific point markers on the image window for reference They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and will be drawn in the corresponding color Point markers assigned to a feature are numbered sequentially in the order that they are drawn on the image Position the mouse where you want to place the marker and click the left button and the marker will be drawn in the image Right clicking on the image or a selected ROI will also bring up the same menus as discussed in Section 2 10 1 95 Andor Technology Andor iQ USER GUIDE 2 10 6 The Define Line Button IS gt 2 The Define Line button allows you to define line regions on the image for analysis They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and are drawn in the corresponding color ROIl s assigned to a feature are numbered sequentially in the order that they are drawn on the image Right clicking on the image or a selected ROI will also bring up the same menus as discussed in Section 2 10 1 2 10 7 The Define PolyLine Button co gt BB The Define PolyLine button a
208. an image to the Windows Clipboard for use in other programs select Edit from the Andor IQ Image Window then Hamamatsu Orca I ER 4742 95 Firewire Copy to Clipboard a SES ES mle i T OL rol E n Live m Snap Add Event Marker File Image List Region view Fast MIF r Copy to Clipboard a gt Edit Event Markers f Add Event Marker Calibration Crop Image Dimension Editor Format Converter Selector las Wavelength Time reer ri Number g range E range Increment Interval s ms A 4 gt 101 0 199 1 00 100 X 2 This is a standard Windows function PROCES mor Monee allowing Pasting of objects in the wl M Automap 100 111 3 321 x e x 3 clipboard into other programs for example word processing presentation or image editing programs 3 The method of pasting images into other programs will vary but is typically accessed from the Edit Paste menu Again depending on the program being used there is also a standard Windows shortcut assigned to pasting CTRL V Please refer to the program s documentation as to how to copy paste and cut objects to and from the clipboard 549 Andor Technology Andor iQ How To Documents A5 15 How to Share and Backup Protocols 1 To share Protocols and Channels between users and systems using Andor iQ the users and or systems must have the same hardware configurations established othe
209. ancel No Acquisition File f Image List Region View Fast MIP le 2 2 mlel The Crop Image Tool is Edit Event Markers s 2 8 2 4 Crop Image Copy to Clipboard Add Event Marker accessed from the Edit Ehron menu Crop Image Dimension Editor Format Converter Selector It is used to isolate regions of interest so subsequent processing and analysis need only be carried out on that ROI When Crop is selected it sets ROI class to a Crop type and then you can drag out a rectangular ROI for extraction Crop works through all image dimensions 74 Andor Technology Andor iQ USER GUIDE No Acquisition 2 8 2 5 Dimension Editor File S Image List Region View Fast MIP le Copy to Clipboard aae um o s 6 The Dimension Editor is Edit Event Markers accessed from the Edit Add Event Marker a Calibration menu Clicking on the a Dimension Editor Format Converter Selector selected item brings up the panel shown below Dimension Editor MER Name MFMONZT2CH 242 16102009 TIF Type 14 bit grey pseudo Intensity 1 000000 lux x 502 0 400000 um x Dimension Dimension Name Resolution Origin 1st 0 4 0 4 Wavelength nm 1 O Te 1 Modify Modify per T ime ms E xposure E vent Modify Modify Modify Modify 0 0 0 0 Montage fe 0 0 0 0 1 NB To edit 1st and 2nd dimension use Y calibration Add D
210. and operates in two modes and has the following key functions Pass through mode FRAPPA acts as an optical relay system with 1 1 imaging orange path in figure above FRAP mode galvanometers are aligned to steer and image laser light from a single mode optical fiber onto the specimen in a point scanning mode Red path shown in figure above Mode switching in lt 5 ms Integrated control with iQ software provides point and shoot and sequenced protocol modes Both allow arbitrary multi region scanning of points lines and 134 Andor Technology Andor iQ USER GUIDE polygons The dwell time of the laser or the length of time the laser is exposed on each point can be controlled from Device Setup Also the same region can be bleached a specified number of times thus allowing accumulated exposure This allows control over exposure control at the point level as well as the region level Under Andor iQ control the user commands FRAPPA to bleach or activate regions of interest with user defined times laser lines and powers Laser switching is tightly synchronized with FRAPPA modes of operation using our proprietary laser combiner multi port switch MPS 3 6 4 What Information Can FRAPPA Reveal Imaging the effects of FRAP or PA in living cell soecimen can reveal qualitative and quantitative information about intra cellular molecules structures and their environment UV uncaging is used to deliver agonists to speci
211. and with a dual disk RAIDO configuration the disk bandwidth is more than sufficient 4 7 1 Dual camera Sequential operation IN this mode of operation iQ can load more than one camera inot a single instance and use them sequentially The Protocol Tree has a Camera Selection keyword which allows the user to specify which camera is going to be used for the acquisition Camera Selection functions in both FLZ and Time oaripaer A Lapse but note that in FLZ both cameras must be Time out in Ext Trig mode triggered from PCU or BOB DO7 Failure to make the Please check cable connection connection between PCU or BOB DO7 and the Camera Ext Trig input will result in a Timeout message as shown right 219 Andor Technology Andor iQ USER GUIDE 4 7 1 1 Camera Selection in Sequential Mode Acquisition Analysis AGB Analysis Calibration Movie Editor Spot 4 gt Status Trt The Camera Selection keyword defaults to Current as shown n Live di n Snap here but a right click Edit on the Save item will show a list of cameras eae Curent which are loaded in iQ and allow Auto Save a h B G Andor iGData indor Clara autolmages ee you to choose one for the current Acquisition Calibration Protocol Select Clara 60Xoi 3 Y TEETE Test Fast j Protocol When more than one camera is 2a Image Test Fast Protocol Type Fast Time A Camera B
212. anges if required When you are happy with the new protocol click the Run button 208 Andor Technology Andor iQ USER GUIDE Note Autofocus and Editing for New Specimens If you have inserted a Autofocus action and Activate interval make sure you initialize the Autofocus hardware BEFORE you Run the Protocol otherwise you will see an error message and warned to initialize again lf you are scanning a new specimen or a new well plate even of the same pattern then you will need to Edit the Scan from the Protocol Tree or Scan Wizard When you get to the field review part of the Wizard you must reset all of the Z values and initialize Autofocus at every field or well Make sure that you have the Device Setup dialog visible and the microscope tab is selected Then click on the Initialize the Autofocus button in the GUI for each field required iQ should indicate if the Autofocus was successful When you click Next Field or Next Well iQ will record the Autofocus setting from the microscope Press Continue Click OK to start protocol Press OK to start the protocol 209 Andor Technology Andor iQ USER GUIDE 4 6 Flexible Protocol Patterns The new flexible Protocol system provides so many possibilities it is not possible to list them all However as you gain familiarity in creating and editing Protocols you will notice Patterns of Actions which recur In this section we illustrate ten Use Case patterns which hav
213. annel then the hardware devices will move to the positions specified for that channel In Live mode the system show a Live image of channel On pressing Snap an image will be acquired after which all post frame events will occur such as all shutters will be closed For more complex image acquisitions you will need to set up a Protocol Before you can do this a valid XY Calibration Sections 3 2 must be loaded and appropriate Channels Section 4 2 and Scans Section 4 3 must be defined 154 Andor Technology Andor iQ USER GUIDE 4 2 Channels and the Channel Wizard In the Acquisition Dialogue Section 2 7 1 press the Channel button Auto Save SR C Andor iQData M Browne R evolution Time Lapse 4utolmages pa Acquisition Calibration Setup Wizard Optovar i ep TO 897_40xwi Channels 7 Protocol Test A Protocol T Channel Manager Image Test Protocol Type Time Series Es Protocol Camera Binning Use Current 4 Camera Binning Use Curren i 1o Wait Allocation Off Run Camera Selection Use Current gp Channel Events fe XY Scan MFx3F Mon Montage Acquisition __New Edt Copy _Delete _ Close fi A Focus A Device Setup Pause Click OK to start protocol Channel Summaries 1 4 Repeat T 50 times 1 min Loop Protocol asi Auto Focus Activate Every 2 times IV Display Image Current Channel EE Repeat XY F Show All red Jo Repeat
214. any dimension the resultant image copied to the Image List However the Selector has the benefit that it does not create a new copy of the data but an access filter which only operates on the specified frames This makes it extremely efficient and fast to create In the above example the image file Plugin Test tif comprising twenty time frames has been sampled using the Selector to create an image file PlugInT est tif_Selector comprising six time frames The Selector operates on the image currently displayed in the Image Window the image details are displayed in the top panel of the Selector dialogue The name of the image created as a result of the sampling process is shown in the Resultant Image Name text box The image name can be defined by default i e ImageName_ Selector or given a unique identification by typing a name into the text box 78 Andor Technology Andor iQ USER GUIDE The range of each dimension to be sampled is selected using the Min and Max slider bars For the example shown the resultant image will contain a total of six time frames The first frame in the series is equivalent to a time of four milliseconds in the original data set the last frame is equivalent to a time of nine milliseconds The separation between frames during the sampling process is determined by the Interval parameter and may be adjusted using the spin buttons The Selector is linked directly to the Image Navigator and hence to
215. ardware Devices The hardware devices included with your system can be set up and Fretocal controlled by pressing the Device Setup button on the Acquisition Pole a dialogue or the Image window tool bar Aun exa So mlelel t lL x rom pd Live N Snap None l Device Setup m Run C en Loop Frotocol W Display Image Channel iiri Store Store As Ph This opens the Acquisition Auxiliary Devices dialogue which contains pages for the setup and control all your hardware devices The page tabs available for a particular system depend upon which devices have been activated for that system using the Configuration Manager in the Andor Bio imaging Program Group Section 2 3 1 and Appendix 2 AAA Andor Technology Andor iQ USER GUIDE Acquisition Auxiliary Devices Fr Rate asso TO Camera Prior DU897_BV 3771 Settings Exposure General Device Info lt lt Sub windo Real EM Gai Window 5122512 Enabled Binnin 3 E H Shutte m No Internal Shutter Pre Amp Gain 5 1 0 Exposure Tim Frame Transfer 30 0 ms Actual 30 000ms Readout ms 32 034 Actual Frame Rate 0 00 Frames to be average Display Every Frame C Display 5 Frames s Free Run Ext Start Ext Trigger Timeout fi 000 ms Fastest Avail Memory MB 185 Camera Statu Camera is working Tips ge Temperature 70 a 2 Note In the above example there is onl
216. arview Deconvolution performs deconvolution processing on 2D 3D 4D and 5D resolution enhancement and reduction of out of focus images of any dimensionality and delivers haze It is therefore aimed at applications where Episodic Average Mask Wizard Field Split Flip Rotate Image Image Filter Rolling Ball Filter Threshold Image ClearView Deconvolution Best Z Image Math Ratio Image Image Image Math Dual Image Disk resolution enhancement will deliver significant benefits This is especially true in live cell imaging and Cytogenetics i 83D 4D and 5D studies of intracellular activity e g translocation signaling and expression li FISH and G band chromosome imaging and spot imaging in interphase cells ClearView uses a measured point spread function PSF to perform constrained iterative deconvolution processing The psf is a fundamental feature of all imaging systems and refers to the spreading or blurring introduced by an imaging system when it is used to image a point object The image of a point object e g fluorescent bead 0 1 um is not a point nor is it constrained to the plain in which the point exists Using this knowledge and the principle of image formation called convolution it is possible to formulate algorithms to reduce the effect of the psf The underlying principles of this technique are outlined in the Clearview User Guide A flow chart depicting the sequence of steps in the Dec
217. at have been previously loaded or acquired into the iQ Following the specification of appropriate source image s ratio mode image background and masking operations the Ratio output delivers floating point images which directly map ion concentrations at the press of a button 378 Andor Technology Andor iQ USER GUIDE 6 11 1 The Ratio Tab The Ratio tool is powerful and flexible and can be adapted to many experimental set ups It provides four different ratio methods four background correction techniques mask creation and mask processing and calibration by either Almers Neher or Grynkiewicz Poenie Tsien equations to deliver calibrated ion concentrations in single cells File About 5 x A given configuration is referred to as a transformation which SOLUCE Background Mask Calibration can be saved and loaded Ratio Source Ratio within M dimensional Image Mumerato SINETSERVER DEMOIMAGESSCA RATIOSFURA TIF Select to from file via the Settings menu for easy re use The Ratio Dimension OF Interes wavelength Time tf Event Numerator 340 Denominator 380 graphic in the dialogue reflects the current transformation The resultant floating point image series are stored to the Andor ImageDisk and can be Ratio two images or image series Ratio two images or image series taking average of denominator Self Ratio taking average of selected range for de
218. ate description into this text box IMPORTANT NOTE You must give each filter position a different name to avoid confusion as the controller uses these names to index the wheel The numeric value i e the wavelength of the filter represented by the selected position should be entered into the Numeric Value text box The Units of the Filter should be entered into the Units text box default is nm When moving the mouse over a filter position in the Wheel dialogue the name numeric value and units assigned to that position will be displayed in a popup The name numeric value and units of the currently selected position are displayed at the centre of the Wheel dialogue Descriptive text can be associated with each filter position to display user information about the filter at the selected position Simply type the required text into the Description text box Press OK to close the Filter dialogue accepting any changes to the settings Press Cancel to exit the Filter dialogue ignoring all changes 468 Andor Technology Andor iQ USER GUIDE Clicking in the lower right hand box of the Filter Basic colors me dialogue opens a colour palette isto A Selecti lour f th lett defini mE im electing a colour from the palette or defining a am custom colour will display the selected filter position ML asa circle of that colour in the Wheel dialogue Fr iE E g E g Define Custom Colors gt gt Cancel A3 4 3
219. ate fields Click on Next Field when you have specified the start and Please ensure image is focussed at each field Z position will be recorded if available want to the end field Press Next Field to select start of the linear feature and prompt for end position x position 15259 3575 Click on Finish to select the last field and y position 2081 0 017 z position 108 33 DN Next Field gt gt Z Position KL LEE 108 33 gt gt gt gt gt gt terminate the Wizard 4 3 5 2 Region and Edge Montage Scan Set up XYZ options The Region and Edge montage scan definition wizards result in a rectangular petal et Ws Setup set of fields to produce an image of a lt Back Newt gt gt feature that exceeds a single Field of Monte go Deanien View In the Region case you can iQ will automatically adjustto the selected calibration Luca R_10X C Linear Specify the start and end of a linear feature millimeters as shown below While in specify the area to scan in Fields or in LEA e An Ta Peras the Edge mode you specify fielas that O j Specify the width and height of th ion PE a o demo ds oe are at the edge or boundary of the centre of the region Region Options Please define the width and height of the region to be sampled Width 3 gt one computes the enclosing rectangle for feature you want to image and iQ Height 3 PES scanning and defines fields
220. ation To get started we include the following check list of actions to ensure proper effective use of Andor iQ These should be completed by the Administrator Installation personnel or key user of the system before general access is provided to others Check List 1 Configuration Manager specify hardware to be controlled 2 Run iQ and test hardware communications boards etc through Device Setup dialog 3 Calibrate optical system um m objective s optivar C mount camera pixel size 4 Run Stage Align Wizard if a multi well or micro plate specimen is to be scanned 5 Create one or more Channels Wizard or Device Setup Create and Edit Scans N O 8 9 Create and Run Protocol s Acquire Images and Data Export and Transfer Images and Data for storage and further use 25 Andor Technology Andor iQ USER GUIDE 2 3 1 Configuration Manager NOTE The Configuration Manager is only available to Administrators To run the Configuration Manager click on its icon in the Andor Bio imaging Folder on the Windows Start Menu AFTER INSTALLATION ASK LOCAL ADMINISTRATOR TO RUN CONFIGURATION MANAGER FOR DETAILS SEE APPENDIX 2 3 1 1 Configurations Hardware Configurations can be created edited and deleted in the Configuration Manager CM The CM interface is shown below with one configuration expanded to show details of device controllers re Kinetic Device Configuration Manager j x F
221. ation Program from which you should open the Software Protection page and select the option Install Dongle Drivers This will open the Rainbow Technologies Sentinel installation Sentinel System Driver InstallShield Wizard E x Welcome to the InstallShield Wizard for Sentinel System Driver The InstallShield R Wizard will allow you to modify repair or remove Sentinel System Driver To continue click Next i Cancel 118 O Andor Technology Andor iQ USER GUIDE Accept the terms and conditions of the license agreement i Sentinel System Driver InstallShield Wizard License Agreement Please read the Following license agreement carefully All Products Gineluding developer s kits Sentinel hardware keys diskettes or other magnetic media software documentation and all future orders are subject to the terms stated below If you disagree with these terms please return the Product and the documentation to Rainbow postage prepaid within three days of your receipt and Rainbow will provide you with a refund less freight and normal handling l Youmay not copy or reproduce all or any part of the Product except as authorized in item 2 below Removal emulation or reverse engimeering of all or arny part of the Product constitutes an unauthorized modification to the Product and is Tdo not accept the terms in the license agreement Installshield lt Back Cancel Select the Cus
222. been installed KlAktuator ocx Filter ocx KlAktuatorController ocx Ic ga CE KICaimController ocx E Program Files Kl CainFilterw heel ocx EJ Andor Bioimagin KICaimMono ocx C be ale KlConix2Motor ocx sx Common Files z KICRIMicroColor ocx C ConfigurationFile4rchive KIDAndorLZRATA ocx CA Graphics KIDAndorRTA ocx CA Images KIDHamamatsu0rcaFW ocx KIDMF ocx KIM arzhauserAxisM otor ocx Kl MarzhauserController ocx KI Prior ptiS canController ocx KIPriorZM otor ocx KlSutterDG4 ocx KI SutterFilterController ocx KSutterFilterwheel ocx KITillMono ocx KI Trigger ocx Selecting the Register Unregister Any ActiveX Component menu item displays the following dialogue Please note the regsvr32 exe file must reside in the same folder as the ActiveX component for the registration process to work correctly select the file to be registered unregistered by defining the correct file path in the right hand panel and highlighting the appropriate file name in the left hand panel The files displayed are determined by the file extension entered into the Filter text box Files are registered or unregistered by clicking on the Register or Unregister buttons respectively Done exits from the Register Unregister Any ActiveX Component menu A2 1 8 2 Registering and Unregistering Plug Ins Selecting the Register Unregister A Plugin Device menu item displays the following dialogue 441 Andor Technol
223. ber of fields is maintained the positions of PF Protocol protocol Frotoco El Image image ane ee E era a eee GE 2C Mil DA z T aoe ca E T n P ocr Insert 3 Interval 792 me fastest Channel Repeat 1 times E I ado Repeat Macros o A Scan Pattern Costar 6 well plate Device Setup E well E ATA 6240 31500 T Loop Protocol o SO xy 2 10219 31500 nese play Image Y 3 6240 13307 0 OD well Store i SI well 3 EA wel4 Store z DA wel 3 LO EP well must visit the field focus the AO seal AT BI Sampling Density 3 specimen at that location and a FA Channel dave test each XY pair are recomputed and the protocol can be subsequently repeated using a revised sampling set Note Autofocus when you want to set new Autofocus values at the new fields you use the microscope tab on the Device Setup dialog to Initialize Cc Autofocus for every field or well as required iQ will indicate if the Autofocus was successful When you click Next Field or Next Well iQ will record the current Autofocus setting from the microscope 189 Andor Technology Andor iQ USER GUIDE 4 4 Protocol Tree Actions Insert Delete and Edit 4 4 1 Protocol Manager Protocols are created and stored with a specific hardware Configuration and they are not accessible outside the Configuration You can use this feature to allow different users to keep
224. ble Acquisition Auxillary Devices i s x for viewing in analysis and image navigation Input Output Set Up o A By default the keyboard Spacebar is used as e a the manual event input But you can also i introduce events from external sources via the Liew fon O Trigger interface The text label associated with pase the selected Trigger input will be used as the description field for each event it creates Before editing the Event Command make sure the Trigger device is setup as described in the Section 4 2 4 with the appropriate text labels to ease Event interpretation at a later date See the panel on the left Py Protocol Furaz aE Image Fura 3 ia To configure the events a Protocol is setup as O Keyboard Events O Opes normal and then the Events command edited as Picospritzer High a shown here Access the Edit Events panel by O Interval 264 ms fastest l a l G5 Repeat 100 times Right Clicking on the Events command in the eg Channel Fura380nm 1 Snap Tree 3 ae Channel Furas40nm 2 232 Andor Technology Andor iQ USER GUIDE Setup Events Keyboard Events M Enabled Spacebar IO Externally Triggered Events When you Edit the Protocol the dialog shown here will be displayed and you can choose to switch on select Triggers to be monitored during this Protocol and off the keyboard events and select the Externally
225. bs Acquisition Analysis AGE Analysis Movie Editar Spot Analysis Status e Window Menus Auto Save E C Andor iQData e boyle F evDSO Adutolmagest 8687 40 o1 Protocol Protocolli Se Image Protocol 0 Camera Binning Use Current Es alee sie i ceo Wat Allocation OFF Camera Selection Use Current F Run Events Move Channel Current F Channel D Repeat T 2O times O ms fastest ES xv Scan af Snap Frame End T y Device Setup C Repeat T 2 times 1000 ms Repeat 2 1 5 um in 16 planes Loop Protocal ig Snap Frame W Display Image Show All Save As a Options 7 Help Andor Technology Andor iQ USER GUIDE 2 5 1 The File Menu The File Menu is used to load images from and save images to disk to specify the default directory for saving Experimental data and also to exit from iQ Image Lis E Settings Open Image Save Image Settings Exit Sek Save Path Image Importer Tl rT Ff images using the Multi Load option dimensional data following the selection of a single file in the appropriate directory Look m a Mu Computer 42 decon testo tif Ea decon testo ti Fi decon testos tif E decon testod4 tif E decon testos tif E decon testo 6 ti E decon testo tif E decon testos ti Ea decon testos tif E decon testio tif E decon testil tif Fil
226. button for a color image throws up the following message Colour Images will analysed as grey Luminencel Intensity thresholds will be set to 0 and 255 Andor Technology 248 Andor iQ USER GUIDE This means that a color RGB image will be analyzed as a grey luminance image The red green and blue components of the image will each be given a weighting by multiplying the pixel intensities by the following values Red 0 299 Green 0 587 Blue 0 114 The minimum pixel intensity value will be set to 0 the maximum pixel intensity value to 255 The data calculated from the analysis is displayed in graphical form The data for each Region Of Interest is represented by a single line the lines being color coded for clarity To zoom in on magnify features on the graph drag out a region with the mouse Position the mouse cursor at the top left corner of the region you wish to magnify hold down the left mouse button move the mouse to the bottom right corner and release the mouse buiton To zoom out again in order to view the whole graph drag out a region as before starting from the bottom right to the top left This zoom facility can be used in conjunction with the analysis graph scroll bar to aid in visualization of the chart The Auto Range function can be toggled on and off by a mouse click When on the check box is labeled with a tick and the Analysis plot is automatically scaled for greatest clarity Mean I
227. camera is connected at an unwanted angle The available corrections are as follows Clockwise 90 degrees and 180 degrees 458 None Clockwise 90 degrees Anti Andor Technology Andor iQ USER GUIDE A3 3 DIO and DAC Card Installation for PCU and FLZ A3 3 1 Introduction Andor iQ Core controls external hardware for Trigger and Event detection PCU while the Fast LZ module controls illumination Monochromator Sutter DG4 and AOTF and Piezo Focus or Z motion These devices require the inclusion of a PCI card into the computer and configuration of the card before use This document gives a brief summary of the correct procedure to follow The recommended digital and DAC boards are manufactured by Measurement and Computing Inc Andor iQ uses two types of board PCI DIO24 or PCIM DDAO6 16 DIO24 is used PCU for TTL synchronization isolation only PCIM DDA0O6 16 is required for PCU and or Monochromator Sutter DG4 and or Piezo focus control A3 3 2 Installing Your DIO and or DAC Card The installation has four steps 1 Shut down PC Insert the PCI card into a free PCI slot Make sure that you use the recommended anti static procedure ground yourself with a wrist strap to avoid damage to the board while handling 2 Install driver for PCI DIO24 or PCIM DDA0O6 16 card Start PC ignore windows found new hardware wizard Insert Andor iQ installation CD into CD ROM Run Setup exe from the installation CD select F
228. can ScanName 3MFMontageZScan FileVersion 2 Centre False 522 Andor Technology Andor iQ USER GUIDE Channel WellPattern Density 0 Calibration XYFields 2 16168 142 16935 022 116 66 YZSTEPO 15499 316 15636 097 112 5 YZSTEPO ZScan STEP6 02 10 Montage Region Overlap 5 Width 570 901960784314 Height 613 622615803816 Units Fields Plates MontageOffsets 9 187 019607843137 201 014305177112 0 0 201 014305177112 0 187 019607843137 201 014305177112 0 187 019607843137 0 0 0 0 0 187 019607843137 0 0 187 019607843137 201 014305177112 0 0 201 014305177112 0 187 019607843137 201 014305177112 0 XYZScan End Region Info Fields Field Width 196 862745 Height 21 1 594005 Region Info Fields Field End Region Info Fields 18 5 RegionData6 0 08 01 2010 10 43 13 AM Class Name Class Colour Position X Position Y Position Z Fields 32768 15981 122392 16734 007695 116 660000 Fields 32768 16161 945921 16720 915052 116 660000 Fields 32768 16355 161608 16734 007695 116 660000 Fields 32768 15981 122392 16935 022000 116 660000 Fields 32768 16168 142000 16935 022000 116 660000 Fields 32768 16355 161608 16935 022000 116 660000 Fields 32768 15981 122392 17136 036305 116 660000 Fields 32768 16168 142000 17136 036305 116 660000 Fields 32768 16355 161608 17136 036305 523 Andor Technology Andor iQ USER GUIDE 524 116 660000 Fields 32768 112 500000 Fields 32768 112 500000 Fields 32768 112 50000
229. can check this with considerable accuracy using the Line Profile Zoom function which is activated by dragging an ROI on the graphic around the edge of interest shown in our example below see white rectangle The graph will Zoom to show the ROI at higher resolution Line Profile Focusss E Fie wiew Edit Mode 1900 yellow 1800 1700 1600 1500 1400 1300 1200 17004 The above picture shows the means of Zooming on the Line Profile intensity edge for precise alignment of the edge position on the CCD chip Line Profile Focus8 i IOl x File View Edit Mode Pere ere 1300 1800 1700 1600 1500 1400 1300 1200 1100 p o vellos The above picture shows Zoomed intensity edges for precise alignment of the Field Splitter and the CCD Note It probably will not be possible to get both lines to cross at exactly the same 353 Andor Technology Andor iQ USER GUIDE location due to physical tolerances in the aperture of the Field Splitter but the best compromise should be aimed at taking into account both the 1 4 and the 3 4 crossing points Post capture alignment in the Field Splitter tool will help to overcome these limitations but there will always be a small residual misalignment which should be evaluated as part of experimental error analysis Once these steps have been achieved the orientation width and alignment are complete Now it
230. can have an impact Despite this effect FLZ FT provides the highest frame rate available 553 Andor Technology Andor iQ How To Documents A5 17 How to Configure Triggered Looping with FLZ In this note we show how to synchronize with an external device via TTL and acquire a series of episodes or events In this case the user wanted to acquire a series of stimulus response signals using the iQ system To get TTL signals in and out of the computer an effective solution is to use the Andor PCU A low budget solution can make use of the Andor Break out Box and a PCI DIO card shown below Acquisition Auxiliary Devices Input Output Set Up Controll Board Bd 1 PCI DI024 x Reconnect PCI IO24 SIPSCKT The card delivers 24 TTL signals but we make use of 8 inputs and 8 outputs 1 To install the card with iQ you can follow instructions in Appendix 3e 2 Then create a Configuration in which the Kinetic Trigger Device and FLZ camera drivers are loaded 3 Start iQ select this Configuration and then in the Device Setup dialog shown above go to the Trigger device Settings tab and make sure that the Control Board is connected as shown above If not selected use the drop down to select the board 4 On the Trigger tab you see Input and Output tabs with corresponding input and output TTL signals shown 554 Andor Technology Andor iQ How To Documents Acquisition Auxiliary Devices Curet
231. cation Off Camera Selection Use Current fogo Events dP Pause Click OF to start protocol gi Repeat T 10 times 15 sec S Repeat Channel Ch1 Ch2 Ho Channels Description Repeat 10 umin 11 planes a Snap End Z di End Channel i End TO This Protocol was built to ensure that functionality previously available in FLZ will function in iQ2 Protocols Note here also the use of the Repeat Channel iterator as a compact means of expressing the order and list of Channels 4 6 6 Multi Channel Time Lapse with Z Stacks Channel first This Protocol is paired with 4 6 5 to ensure that functionality previously available in FLZ is functional in iQ2 Time Lapse In this case we simply change the order of Py Protocol Test Case O Channel First so Image Test Case First i Frotocol Type Time Series ai Camera Binning Use Current Wait Allocation Of Camera Selection Use Current i Events dP Pause Click OF to start protocol Gi Repeat T 10 times 15 sec Sere Hepeat T0umin 11 planes Repeat Channel CAI Ch2 H Channels Description it End Channel al Snap End 2 W End TO 213 Repeat Channel and Repeat Z so that the Channel now is the first to iterate These Protocols are important for flexible control and optimizing acquisition speed depending on which hardware is slower Z scanning or excitation and emission wavelength switching Andor Tech
232. cccsssscssccccsscssssssscccsssssssscssccsssssees 564 AS 20 HOW t Create Protocol Template sciyecivscccscccevyavessoasesecosesesceveecenessavsacecsoasecedocecutenseepesscacuueeueversecetosees 566 A5 21 Configure iQ with Lumenccor Light Engine cccccccsssssssscsccccsssssssssccccscesssssssccccsccsssssssccsssseees 572 5 Andor Technology Andor iQ USER GUIDE 6 Andor Technology Andor iQ USER GUIDE eo TECHNOLOGY ANDOR 1 Welcome Andor Technology Andor iQ USER GUIDE 1 1 Introduction Welcome to Andor Technology s scientific imaging software Andor iQ which is focused on multidimensional image acquisition iQ provides processing and analysis functions closely associated with acquisition and now partners with Bitplane Imaris as the preferred visualization and analysis platform Andor iQ has been developed with multi dimensional live cell and time lapse imaging in mind and therefore has many powerful features not found in conventional image acquisition products but is equally at home capturing data from fixed specimens Since iQ can control a wide range of experimental devices including cameras EMCCD Cooled CCD illumination controls e g laser combiners with AOTF shutters filter wheels etc motorized stages and microscopes sophisticated acquisition protocols can be easily setup and utilized The current range of supported devices is listed in section 1 8 Since this range
233. ccess to DSD turret changes is especially convenient in the Andor design and can be performed while the DSD remains attached to the microscope as shown A3 10 1 USB Connections and iQ control In order to control the DSD it is necessary to have the unit powered and a USB connection made to your computer The screen shot over the page shows the DSD Settings tab which is accessed via the Device Setup dialog In the settings tab you select Device 1 or if you have no device but want to explore the interface you can select Demo as shown You can also specify which Turret is currently selected 491 Andor Technology Andor iQ USER GUIDE Acquisition Auxiliary Devices Current N Live N Shap Control Settings Blot ne r Status Connected On DEMO Device Select DEMO T MV Reconnect at Startup Reset Turret Select Curent Turret GFP DsRed Once the device is connected you will have control via its GUI for manual control The settings can be stored within iQ Channels to ensure they are re used in Protocols for multi dimensional acquisition The GUI controls are shown below Note there are only two primary controls The first is the combo box which selects the position of the internal filter wheels The figure below shows the blue excitation filter center 444 nm for GFP selected and an equivalent green Emission filter will also be selected some systems have multi band excitation filters
234. ces in these two examples For details on how to import pre existing Configurations refer to section 2 3 1 4 of this User Guide 151 Andor Technology Andor iQ USER GUIDE To build a new Configuration select devices to control from the lists shown in the Configuration Manager starting with the camera driver Check the boxes against the selected devices as shown in the screen shot below r c Device Configuration Manager c Device Configuration Manager 152 Edit an existing configuration lt lt Back Select the devices fram the list t Next gt gt Available Auxiliary Devices Andor MicroPoint Device Edit an existing configuration File Edit Settings Tools About Exit File Edit Settings Tools About Exit lt lt Back Next gt gt Select the devices from the list that Available Auxiliary Devices _ Olympus IX81 B 61 Device Andor Mosaic Device JCRILCTF Device _ Andor U YPulsed Laser _ Leica DM l x000 Micro Macroscope O TIILMIC Device _ Ludl Stage Device Zeiss Axioplan Device l Lud Filter Wheel Device _ Kinetic Aktuator Device v Piezo Device _ Andor APZ Device _ Mad City PXY Device v Andor Laser Combiner Mad City PZ Device ASI Stage Device ASI Filter Wheel Device _ Atto KIAtOoAIC Device Cairn Filter Wheel Device L Cairn Monochromator Device _ Marzhauser Stage Device _ Kinetic Monokromator Device _ Nikon IntensiLight Device
235. cian Sobel Prewitt Sharpen Median Dilate and Erode Enter the number of repeats in the Repeat textbox and press Preview to see a preview of the selected filter and repeats in the Image Filter window Press Snap to load this single image into the ImageDisk and iQ s Main Window Press Run to use this filter to operate on all images in the sequence and create a new image in the ImageDisk 356 Andor Technology Andor iQ USER GUIDE Press Close to close the Image Filter window Shown below is the effect of these filters on the test image although some are difficult to reproduce in print PDF Shown also are the 3x3 kernels for each filter Original Average to smooth random noise Lowpass to smooth random noise Highpass X Andor Technology Andor iQ USER GUIDE Highpass Y 2D Laplacian edge detection Sobel edge detection Prewitt edge detection Sharpen Median Andor Technology 358 Andor iQ USER GUIDE Dilate to expand bright objects or connect pixel groups left below Erode to reduce bright objects expand dark objects right below 359 Andor Technology Andor iQ USER GUIDE 6 6 Rolling Ball Filter Process Analysis Plug Ins Help The Rolling Ball Filter can be found in the Process menu in Pec Averacs Field Split Flip Rotate Image Image Filter Rolling Ball Filter Threshold Image ClearView Deconvolution Bes
236. cified range for red and blue but NOT green will be colored magenta on the image When the parameters in the Settings dialogue have been optimized press OK to close the dialogue accepting the specified values Pressing Analyse will perform the analysis on all regions defined on the image using the ROI tools if no regions have been defined the analysis will be performed on the entire image The results calculated depend upon the statistics selected on the Options page Section 5 5 1 The data calculated from the analysis is displayed in the table Each row contains the statistical data for one user defined region of interest The following data is calculated for each of the filtered planes specified in the Multi Wavelength dialogue on the Grab page e g for each of the red green and blue planes of a RGB image This data is displayed on the Channel page Mean the mean intensity of the pixels for the red green and blue channels Peak the peak intensity of the pixels for the red green and blue channels SD the standard deviation of the pixel intensity for the red green and blue channels 256 Andor Technology Andor iQ USER GUIDE Area the number of pixels within the region within the threshold limits specified in the Settings dialogue for the red green and blue channels Area the percentage of the area within the threshold limits specified in the Settings dialogue for the red green and blue channels I
237. col N Aun Channel un Pause Click OF to start protocol oi Interval 132 me fastest I Repeat 1 times BO Scan Device Setup Ha N im Loop Protocol W Display Image Store Store As E 187 For the example shown adjustments to the first XYZ position can be made using either the Move To or Record Current commands Note Autofocus when you edit fields adjust Autofocus at the new field the microscope tab Then click the Initialize the Autofocus button in the GUI iQ will Use Device Setup dialog indicate if the Autofocus was successful When you click Record Current iQ will record the current setting from the microscope An corresponding to a Z series is shown expanded Protocol Tree on the left Clicking on Repeat Z with the left mouse button and then clicking the right mouse button displays the Edit and Insert functions Pressing the Edit button accesses the Scan Manager and allows the Z scan settings to be modified together with the channel specified for acquisition Andor Technology Andor iQ USER GUIDE 4 3 8 Focusing During a Scanned Acquisition The microscope hardware Auto Focus feature which corrects for focus drift during the execution of a Protocol has been explained However if this hardware is not available you may find that during the execution of a scan or single field imaging it is necessary to adjust
238. compare any two cell parameters can also be plotted The scatter plot chart function is accessible via the Scatterplot button or from the Plots option on the Data Review menu bar The plotting of histograms and scatter plots is discussed in Sections 5 7 6 3 and 5 7 6 4 respectively 5 7 6 2 Show Spot Gallery This is a visual method of viewing data in which the spots previously analyzed are shown as an image gallery Clicking on the Show Spots button opens an Image Review window which displays the quantified features e g cells or cell nuclei in the form of a gallery There are up to 8 parameters displayed for each cell or cell nuclei as specified previously in the Review dialogue 292 Andor Technology Andor iQ USER GUIDE Image Review File Plots Data NA e e e o jo a O ee ee eee No V Area Perimeter f MaxChord Shape TInt Int Meanlnto f LogInt Int Mean Int Area 62 1261 000 166 000 50 000 fo 575 116156 000 92 114 11 663 fo 073 Image C TEMPSCOLONA AVI Colona Enclosing region 470 199 506 242 Time 15 Histogram Scatters ol Total Spots Clear Selection Included Spots 99 Print Spots vox X Cancel To display the data associated with a particular feature click the left mouse button on the image to update the parameter text boxes To remove an image from the data double click on the image The image will be tagged with a
239. computed by clicking on the associated radio button at the bottom of the dialogue AZ The distance between adjacent image planes To compute this value you should enter the number of planes and then move to the final position of the Z 164 Andor Technology Andor iQ USER GUIDE Series using the Z Motor As you move the Z Motor the current position and computed AZ value are displayed Pressing Select End will accept the current position as the End position the Z coordinate at this point and the corresponding AZ value will be recorded There is also a right mouse option in the protocol tree which allows two Channels to be acquired with a Z offset between them This may be desirable in phase contrast versus fluorescence channel or between two different fluorescence channels depending on the localization of the fluorophores Number of Planes The number of slices in the Z series To compute this value you should enter the AZ value and then move to the final position of the Z Series using the Z Motor As you move the Z Motor the current position and computed Number of Planes are displayed Pressing Select End will accept the current position as the End position the Z coordinate at this point and the corresponding Number of Planes will be recorded Z Positions The coordinate s in the Z Series To compute this value you should enter values for AZ and the Number of Planes Pressing Select Start will accept the current position a
240. configuration Orca Ludl e Y Created By Mark Browne O Date Created 2 28 2005 10 32 24 AM o Date Last Modified 2 28 2005 10 32 24 AM 4 Acquisition Device Hamamatsu Orca VER 4742 95 Firewire a Auxiliary Device 1 Ludl Stage Device a Auxiliary Device 2 Ludl Filter Wheel Device 434 Andor Technology Andor iQ USER GUIDE Created By The name of the Administrator who was logged onto the Local Machine when the Configuration was created Date Created The date the Configuration was created Date Last modified The date the configuration was last modified Acquisition Device The Camera Device used in the configuration The Camera Device Driver OCX used by the Camera Device can be seen by expanding this part of the tree Auxiliary Devices The Auxiliary devices that are contained in the configuration are listed Auxiliary OCX Files The Auxiliary Devices Drivers OCX s used by the Auxiliary Devices can be seen by expanding this part of the tree This is useful for troubleshooting A2 1 3 Creating a New Configuration 5 Device Configuration Manager sim Edit Settings Tools About Exit view Configurations ard New Configuration Current Configurations cal E Pressing the New button or selecting the New Configuration item from the Configuration Manager menu launches the Configuration Wizard 132 O Andor Technology Andor iQ USER GUIDE ua Device Con
241. cro oD End symbol adjacent to each item in Scan the Protocol Tree using the left Device Setup mouse button T Loop Protocol W Display Image Store The example scan illustrated Shore Ag comprises 4 XYZ locations for each well in a 6 well plate The individual XYZ scan positions are displayed for the second well Expanding the Protocol Tree and clicking on the appropriate XYZ position using the left mouse button can select any set of coordinates in the scan pattern An example is shown below for the scan coordinates XYZ 5 in the second well Clicking on the highlighted coordinate set with the right mouse button displays a pop up with two commands Move To and Record Current 185 Andor Technology Andor iQ USER GUIDE Sige Protocol protocal Protoca By moving the stage and clicking on eg Image imageN ame Pratecal RA Protocol Type Scan the Record Current option USING the off Scan Multi Well Systematic Sampling 2 a Centre on Registration 7 dave test nn left mouse butto n th current n Pause Click OK to start protocol Sheet a Interval 1056 ms fastest ELS Repeat 1 times a coordinate position is updated in the agat Repeat Macros i ap Scan Patter 6 well costar Device Setup list and permanently reco rded with i well wa G Loop Prato the scan Hiii E ijik r E ERT R 2 ms i m
242. ct Point tool e vee oe ils dA 130 Andor Technology Andor iQ USER GUIDE lt lt Back Next gt gt Cancel Stage Calibration The stage will now scan to check that the camera is parallel to the X axis Rotation Error Degrees Error Tolerance Degrees 5 54 F Automap 1000 268 294 z0 x e wl a In this final step of the Stage Alignment Wizard you will see in the Image Window that iQ moves the stage from the left to the right snaps a frame and then computes the feature s position Each move and compute generates two coordinates which correspond to a move in the X axis of the stage From these coordinates the relative angle can be computed iQ displays the computed angle in Bold red characters Below this you can edit the acceptable error for your purposes The default is set to 0 5 degrees which is our recommendation for good performance The quality of Montage or mosaics may be especially susceptible to large errors gt 1 degree in the angle In order to change the rotation error angle simply rotate the camera and wait for the next computed angle to be computed and displayed Go on with this process until the rotation error drops below the error tolerance Click on the Next button 131 Andor Technology Andor iQ USER GUIDE Note After you click Next iQ moves the stage around automatically identifies and measures the feature movements in order to ca
243. d Spot Region Color this enables the user to alter the color of the bounding 82 Andor Technology Andor iQ USER GUIDE rectangles used to identify features analyzed during Spot Analysis A comprehensive discussion of Spot Analysis is provided in Section 4 10 Options this allows the user to change the line width for drawing regions as well as the bitmap for drawing user defined points 2 0 5 The View Menu e Paula drosophila PBC_VerticalFlip_Rotate180 16 Bit 642 0 107 x 1020 0 107 4D File Edit Image List Region RYSa Fast MIP IK VIIA on OIS T i mal O ro L AutoMap Toolbars Z v ToolBar Image Background v Navigator Hello VA Center Image Status Chart Image Strip View 4D View 3D View Montage Kymography Orthogonal Slice View The View menu provides all the necessary tools for the display of images and features in the Image Window The LUT item allows the selection and editing of look up tables Selecting the Mapping or AutoMap items accesses the necessary tools for the adjustment of image contrast and brightness Please refer to Sections 5 1 1 and _ 5 1 2 respectively for further information Image Background opens the image background color options The Image Background item enables the background color of the image to be changed in order to improve visualization of image features Center Image when checked locates the current image view in the cent
244. d bar shutters in the Device Setup dialog shuttere Experiment Action Current Current Open Closed Open Closed_ Closed Open Open Closed Llosed Open the Shutter will not move The experiment action for the shutter is selected from the drop down list the Shutter will be opened immediately before channel acquisition and remain open until another channel closes it the Shutter will be closed immediately before channel acquisition and remain closed until another channel opens it the Shutter will open at the start and close at the end of the acquisition of each channel of the image the Shutter will close at the start and open at the end of the acquisition of each channel of the image Press Next to continue Exposure General Device Info cc Sub Window Window 512 512 Binrmg lv Y lx v hutte No intemal Shutter Frames lo be averaged F te ET J Display Contot f Display Every Frame C Display 5 Frames s Real EM Gary Enabled al z Pre Amp G ain E 10 Exposure Time Frame Tranches tJ Actual 30 000rms Readout mst 32 034 Actual Frame Rate 0 00 2 Finally you will be prompted to configure your Camera which in this example is an Andor iXon The Wizard camera control panel is shown left Deeper levels of camera settings are treated as global and not recorded in the Channel Pressing Live will display a live
245. d Intensity _ Integrated Intensity Per Unit Area Return To Graph Channel Selection My euen Solid Biz Green 2 Dah l 63 Andor Technology Andor iQ USER GUIDE 2 6 3 Movie Editor The Movie Editor is used to create animation sequences from frames of interest which can be subsequently saved as AVI Movies Acquisition Analysis RGE Analysis Calibration Movie Editor Spot Analysis Selected Image 1 Path as 4 bl 2 2 me SOEVELOPMENT TEST REPURTSSTEST _ IMAGESACALCIUMAFURA TIF 25 Animation dim Wavelength Min Frame Tot frames 2 Rate 1 E i i Out nee ER Save List E Load List Animation Dimension roa Save Listas Movie Movie Scaling Factor a Delete selected Images Increment M Multichannel w Set Frame Use Current Region C Play List Save Overlay Fi alk 4 Image List xo penere r ou os oc Ress tin Saas Va fayvelength The use of the Movie Editor is described in Section 5 8 To get started select this dialogue use ImageList to show an image in the Image Window it will then appear in the list Use Set Frame option to edit parameters and Play List to preview Then Save List as movie to save window s AVI 64 Andor Technology Andor iQ USER GUIDE 2 6 4 Spot Analysis The Spot Analysis Tool is used to quantify the morphology density and intensity of discrete objects Further details regarding the use of the Spot Analysis
246. d a constant value which produce a destination the Z dimension for extended depth of field and in the T dimension for highlighting feature tracks Max and Min a Dual Image Disk Encoded projections MEP s are useful in Z for topographic projections and in T for spatial development mapping Load Save Monadic Processing File About When Selected the Monadic Processing i Source Back round Mask window Wwill open Operation Sum Operation Source Image The user can Load and or Save Settings ZADEVELOPMENT TEST REPORTS TEST IMAGES CALC Select by selecting File on the Menu Bar Dimension Of Interest Select About to view product information o d a Source Background Mask Primary No Mask Background O 370 Andor Technology Andor iQ USER GUIDE 6 10 1 The Source Tab S OUNCE Background Mask Differentiate Maxinurn Projection Minimum Projector Maxinunn Encoding Operation Minimum Encoding Delnterlace Uperation Source Image SDEVELOPMENT TEST REPORTSSTEST IMAGESACALO Select Dimension OF Interest Wavelenath Hange Dff al W 6 10 1 1 Operation Select an Operation from the Dropdown list as shown Sum This is used to produce a resultant image whose pixel intensity at any coordinate is equal to the sum of pixel intensities of the input image in the specified Dimension and Range of the selected n Dim For more details of modes of operation
247. d by Windows User Status Administrators are able to run the Configuration Manager and change hardware settings while all users have permission to Run iQ create edit and execute instrument control and image acquisition Protocols If the ImageDisk folder is set to Private the ImageDisk folder name is based on Windows logon name This offers data security and privacy but only if usernames and logons are used correctly It is important therefore to ensure proper use for this mechanism to function robustly lf MUA is OFF all users have full access to hardware settings and probably share the same Windows logon In this case multiple users can be managed in a slightly different way If they share a hardware configuration they can control the same hardware but under different names so that their settings and protocols are stored independently In this case the ImageDisk is Shared by all users and so is less secure but it can work well in multi user environments when users clean up and save their own data after each session OR a robust policy of the ImageDisk will be deleted every night at midnight with Windows automation routine executing a scheduled clean up 21 Andor Technology Andor iQ USER GUIDE 2 1 1 Running iQ While Logged onto a Domain Some customers may wish to run iQ on a computer while logged onto a remote domain In this case Windows grants limited access to certain areas of the local machine Therefore it is very
248. d inside itself but may be dragged within another repeat loop so long as it is not a child of the loop being dragged Channel repeat should not contain any Move Channel items Dragging a Move Channel into the loop is not permitted Dragging the end node around a Move Channel is permitted to allow an intermediate state while editing the tree but you will be warned that this will cause undefined behavior Due to the complexity of the XY scans these repeat loops cannot be dragged doing so would invalidate the structure of the scan The end nodes may be dragged internally to allow items to be accepted into and rejected from the loop 217 Andor Technology Andor iQ USER GUIDE 4 6 13 Irregular Protocol Structures The flexible protocol tree allows a user to create very complex acquisition sequences In order to manage the data sets more easily iQ breaks the image dataset into regular channel based sub images These are joined together into a single image using what we refer to as a Constructor The Constructor allows the irregular data set to be managed as a single object Each sub image is visible within the Image Manager they appear below their associated constructor Whenever possible iQ will try to allocate a single regular data set in which case there will be no constructor 4 6 14 Flexible Protocol Conclusions We hope you find that flexible Protocols in iQ2 allow you to carry out new and exciting experiments which were no
249. d part way through a reconstruction Note the progress information on the bottom Status bar which provides a processed frame number count and a total frame number count in the dataset Before iQ starts the reconstruction it allocates a destination or result image on the IlmageDisk To do this it estimates how large the result will be by looking at the meta data and computing the result X and Y dimensions from the field data and scan settings If the X or Y dimensions exceed 4096 pixels then iQ will subsample the image to ensure it fits within this range The reason for this is that Windows has trouble rendering images that exceed 4096 pixels in X or Y and performance is greatly reduced So we took this decision for performance reasons You can choose the sub sample option before reconstruction if you want to control the result When the image reconstruction Is finished you can click on Show to send the image to the iQ Image Window where we will now see below the Montage dimension is no longer available but the reconstructed image retains all other dimensions V Multi Wavelength um Frame Number Minrange Maxrange Increment Interval s ms a a potes peee nemo met aja idol e p MEZEG SetMin Set Max o 1003 1043 333 Andor Technology Andor iQ USER GUIDE 5 13 FRAP Analysis The FRAP analysis has been provided to allow extraction of quantitative values from FRAP experiments using semi quantitative curve fit
250. d so that the specified number of fields lies within the well Fie Image List Settings QC Wizards Process Analysis Plug Ins Help Setup Wizard The following step allows you to review the Set up XYZ options position of the computed fields and modify them at will Select Z Device Device Setup lt lt 8 ack N ext gt gt Cancel Note Autofocus when you want to record Multi_Field options _ and Autofocus setting at each field make sure Select Number of Fields per well Press NEXT FIELD to select position and move to next field that you have the Device Setup dialog visible Press PREVIOUS FIELD to select position and move to PASE and the microscope tab selected Then Please ensure image is focussed as z position will ee di ii initialize the Autofocus button in the mareou fell Next Well gt gt Bas fee microscope interface iQ should indicate if the y position C t Field 1 coto U s s T Autofocus was successful When you click mg ext Field gt gt on Next Field or Next Well iQ will record the Kc lt lt lt D gt gt gt gt gt gt current Autofocus setting from the microscope 173 Andor Technology Andor iQ USER GUIDE 4 3 4 2 User Defined Fields pa Sy a E E The stage will move to the top left quadrant Setup Wizard of the first well Enter the number of fields Setup x2 options gt for each well Channel CH1 Z Using
251. degree and then indicate on the radio buttons whether the image moves in X or Y This allows iQ to establish which axis is under control for the following steps Return FRAPPA to the Imaging Angle to the calibration value before proceeding Then click Next 138 Andor Technology Andor iQ USER GUIDE FRAPPA Calibration View Setup Wizard FRAPPA Calibration Set up FRAPPA alignment and calibration settings lt lt Back Next gt gt FRAPPA Calibration Point 0 142 198 0 2 0 1 m 1 a a 0 1 oint 2 0 4 oint 3 a a Si 0 4 oint 4 450 460 0 9 0 8 Light Path 0 899 0 799 ie Pos i i i alibration Poin i Delete Last Point Bleach Time and Channel FRAPPA __BleachSample ch Sa __BleachSample 1000 Ae NE if you Zoom the image please reselect Point tool 4 ESEJI M Autom map 1000 2421 1042 IN fe xo A s 6 The FRAPPA Calibration panel allows you to calibrate how the FRAPPA angles translate to camera pixels This ensures that interactive FRAPPA operations can be executed at the correct position Start by setting the X and Y values to the Scan Centre values recorded in the calibration label Note that if you are Editing a FRAPPA calibration then the Wizard should reset the center values automatically Now with the specimen in focus select a suitable FRAPPA channel and click the FRAPPA button If the system has been pre aligned you should see in the image
252. dent Statistics Options MM Automatic Directory Selection Group Analysis Greys Per Second Background Correction COPRUR CREO ER UR TRSR ES DOUS USOS DOUS OS DOUS NS OU DOU CS DO SONS OS TEES CR TRO AS OS DOUS OSOS SUS O UU SS CS TREO E TOES COROT ETO EE COTTE CERO T CETTE TEE COCO EERE TOTO SECO TCE OE TOROS errr Mean Intensity Peak Intensity Integrated Intensity Intensity Ratio Chromaticity Delete ES Save The functionality relating to the checkboxes for Automatic Directory Selection Group Analysis Greys Per Second and Background Correction have been discussed previously in Section 5 5 1 Statistics available for selection in RGB Analysis are as follows Mean the mean intensity of the pixels Peak the peak intensity of the pixels SD the standard deviation of the pixel intensity Area the number of pixels within the region within the threshold limits specified in the Settings dialogue 264 Andor Technology Andor iQ USER GUIDE Area the percentage of the area within the threshold limits specified in the settings dialogue Integrated Intensity the integrated intensity within the threshold limits specified in the Settings dialogue Integrated Ratio the ratio of the intensity of one color component compared to the intensity of another component e g the RedGreen Integrated Ratio XI Red I Green This statistic is only displayed on the Coi
253. dow This may take a little time as the splitting process will be propagated throughout the other image dimensions such as time and Z and even multiple fields A progress bar on the top of the dialog will show the stage of the processing 6 3 3 Tips on Setup for a Field Splitter with a CCD Camera Using iQ Refer to the manufacturer s literature for specifics In this example we use the DualView as our example Attach DualView to iXon or other CCD camera Go Live and refer to the 5 Step Alignment procedure described by Optical Insights Step 2 Orientation To help establish the alignment of the Field Splitter with the CCD imager you can use the Analysis Line Profile in iQ Draw two lines in the horizontal direction Spanning the width of the image Use the Right mouse Edit menu to adjust the individual Line details and ensure that they are aligned and span the image Make sure the X1 0 and X2 511 or whatever the maximum size of the sensor and note that the Y1 and Y2 values will be identical for a horizontal line In our example the Lines are 512 pixels long and cross the edges horizontally If the aperture edge is oriented orthogonal to the CCD chip then the two line profiles should show coincident transition from black no light to grey light at the edges provided they have the same X1 X2 coordinates Andor Technology Andor iQ USER GUIDE This picture shows the aperture image Live on the iXon camera ready for adjustment of
254. e Trigger Out Output a Hi or Lo TTL level on a user selected bit from 8 TTL outputs The digital pattern is shown in the Tree as two groups of four binary patterns XXXX XXXX corresponding to the TTL outputs DO7 4 DO3 0 The hardware TTL outputs are handled by either the Andor PCU100 series or the Andor BOB 100D or BOB 100A interface modules These outputs can be used to synchronize iQ execution to external processes and or control other hardware For example many perfusion systems have external TTL inputs for valve control and electrophysiology apparatus has external TTL trigger inputs Note that you can include TTL Trigger Out actions as part of a Channel as Preframe and Postframe actions If you use Triggers you can set Transition and Duration delays of TTL pulses in the Device Setup Dialog Return to Table Wait Allocation state instructs the Protocol how to handle image allocation on the ImageDisk If state is OFF iQ will start execution of the Protocol while Allocation proceeds Allocation is usually faster than acquisition 500 image file allocations per second are not uncommon but can impact performance To minimize starting delays leave the state OFF especially if your Protocol captures many frames and you do not have high performance demands 201 Andor Technology Andor iQ USER GUIDE If state is ON iQ waits for Allocation to complete before continuing If you need high performance then the ON state is preferr
255. e 2 Select the Andor Software page and click on the software that you wish to install following the on screen prompts The installation creates a program group called Andor Bio imaging and program icons are added to this group To run a program simply double click on the appropriate icon 3 If you are installing an upgrade product uninstall the previous version first Use the Control Panel Uninstall feature Your Protocols Channels and Scans should remain valid and in tact 407 Andor Technology Andor iQ USER GUIDE ANDOR iQ Appendix 2 Configuration Manager 428 Andor Technology Andor iQ USER GUIDE A2 1 The Configuration Manager A2 1 1 Global Configuration Settings When Configuration Manager is first run there will be no Configurations and you will first be invited to setup the Global Settings These allow you to select parameters that will affect User Access level control and ImageDisk configuration c Device Configuration Manager File Edit Settings Tools About Exit Global Configuration Settings Done Multi User Access When you select this feature two access levels are available when running Andor Bioimaging Software IV Multi Level User Control OWN These two levels are determined by local system settings See User Guide for Details Administrators FULL Access Other Users Full Access to Wizard NO Access to Hardware Settings Image Disk Configuration ImageDisk Path C Virtual Ima
256. e processing and analysis of data from one or more cameras In multi camera systems the images may be combined for a variety of reasons These can include comparison or combination of imaging modalities e g TIRF and confocal DIC and fluorescence or as a processing steps to characterize some property of the specimen e g FRET or ratio imaging Andor has developed a flexible optical solution to split information between two cameras The dual port camera adaptor DPC shown allows dual wavelength or polarization imaging depending on which optical components are included in its modular optical cassette system The procedure that follows can be applied to images from other optical configurations but will be described from the perspective of the Andor DPC For accurate data to be acquired from a dual camera system it is important that the magnification orientation and XY position of the cameras match each other as closely as possible Assuming the optical paths are matched then we can use software to optimize the pixel to pixel match On the other hand if the images are of disparate linear resolution on the grounds of binning or pixel size they can be overlaid for visualization but would need to be re sampled for pixel to pixel processing Image re sampling is not covered here 119 Andor Technology Andor iQ USER GUIDE In the following description use of two identical cameras is assumed The process described here is
257. e C Auto PeAmeGai 500 and then Lo TTL 0 again to start the a aa Burst We use Move To make sure the aa pete f devices are in position before we enter FLZ iene cates oe oe and when the FLZ Burst is complete we ee a ae E return to the Protocol and iQ sends a pulse i T c De Tasan T mi High Lo from Trigger Out 0 The pulse T e width was set to 10 ms above Tempeh 70 E e 8 In this case the Camera above is setto Fast LambdaZ mode Because we use the Current channel all the settings in the Device Setup dialog are used for imaging 556 O Andor Technology Andor iQ How To Documents 9 Now let s take a look at the FLZ Experiment tabs to see what happens in the FLZ Burst as shown below Using the Burst control Edit settings allows us to specify the number of Bursts 100 and the Burst interval to be zero elk Burst Control Bright Field Imaging Control Burst foo DO M Enable Interval 0 000 s l Count 100 Int s 0 000 Z O Position After Burst No Bright Field z L Control 0 WV Enabled Move 1st Number of Lambdas 1 L EnabledL M wi E I Enable Current L O ES o Burst And Bright Field Control Camera Trigger DUSS7_BY 4707 Settings Experiment This window is disabled for experiment Sequence gt gt Burst and Bright Field Control 0 00 Excitation WET T Crt maT oe 13333405 i Emission Bo l m Move
258. e Copyright Andor Bioimaging Divizion 1993 software and contact details for Strelley Hall 100 Southcenter Court Strelley Suite SOU Andor systems Division Nottingham Morrisville MGS BPE UE NU 27560 USA tel 44 175 906 1354 tel 41 919 467 O60 fax 44775 906 1353 fax 1 915 467 0608 e mail Infotkineticimaging carn e mail info ustakineticimadging com WAAL andorig com 55 Andor Technology Andor iQ USER GUIDE 2 6 Function Tabs 2 6 1 Acquisition The Acquisition tab enables set up and execution of experimental Protocols 56 Andor iQ 2 2 Andor Camera File Image List Settings Wizards Process Analysis Y Open Plug Ins Help Current Acquisition Analysis RGE Analysis Movie Editor Spot Analysis status e Auto Save Ic Andor D atase boylesAevOSO 2 4utolmages i 66 7 4001 Protocol Protocol Se Image Protocol ab Camera Binning Use Current T wait Allocation OI E Camera Selection Use Current E Events ob Move Channel Current Repeat T 20 times 0 ms fastest gl Snap Frame End T Repeat T 2 times 1000 ms lls Repeat 1 5 um in 16 planes a 2 Snap Frame Andor Technology Protoco Es Protocol O Aun a Channel HA YY Scan ae Device Setup Loop Protocol W Display Image F Show ll Save As Ps Options Help Andor iQ USER GUIDE 2 6 1 1 Status Box The Status Box di
259. e OLC can be used to monitor the intensity in one channel selected from the setup dialog shown below or the Ratio between two channels in a multi channel Protocol Pe Protocol Furaz O Image Fura E Protocol Type MC The OLC will be inserted below the E a Snap command as shown in the Fura oi Interval 264 ms fastest c CO Repeat 100 times ratio protocol left Each time a frame or Be Channel Furag 0nm 1 E l Snap frame pair is acquired the OLC will be fl Channel Furad4Qnm 2 updated anap OnLineChart Ratio Numerator Furas40nm Mean Channel Channel Name I I 1 I I I 1 I PESEE N a aana ke a Se aaa a aea aae a Aa a E ee a a a S aa Cores a aa AA a NN moa a e a Pe ead Roar vow AA a pas A sea PA a a A ads A poi Women via a Aa a AA AA a ala I I I I I I I I I I I I I I I I I I I I I Ratio Channel This plot can also be printed from the File Menu The OLC Window can be resized and placed conveniently to avoid visual clutter A favorite location is below the Image Window and made fairly small in height The OLC will remember its size and position throughout the current session 236 Andor Technology Andor iQ USER GUIDE ee ANDOR ba TECHNOLOGY 5 Analysis Visualization 237 Andor Technology Andor iQ USER GUIDE 5 1 Image Contrast and LUTs Region Fast MIP LUT jaja _ Mapping AutoMap Toolbars Image Background Center Image
260. e Poly line tool and draw some zig zag lines to mark out the contraction and protrusion of the cell 317 Andor Technology Andor iQ USER GUIDE membrane according to your judgement We call this tracing the time profile The figure is not a good example of this process it is included for illustration purposes only 11 When you have traced the time profile of interest click the Tracking button and the data tab will display information as shown below save Tracking Data Tracking Data Lamella Mode Cell 249 247 Line 0 labelled 10 Line Angle Length Ratefum min Dur rin Displum 19 4 a408 1 7E5 0 0003 2 163 51 37 3 06E5 00001667 51 3 12 46 06 26265 O 000166 47 4 165 4 54 25E5 O0002 46 F 11 7 5413 2891E5 00001533 pa B 17 50 64 3 7565 0 0001333 50 i 3 46 36 5 36E5 00001 36 12 The rate and displacement have both positive and negative values positive being protrusion and negative contraction 13 You can repeat this process for all of the lines in the source data Each line will deliver a data set like the one above showing protrusion and contraction rates and will be recorded for a Cell and the cell is identified by the Cross coordinates 14 There is one known problem with Lamella mode This does not cause a crash or a malfunction but does create an error message whenever the Right Mouse Delete or Delete All operation which is used This is the only way to rem
261. e been imported step 3 You will see in the Windows Explorer view on the next page that a folder called ScanFiles exists under Configuration You must now create a new folder called ScanFiles2 and copy here all of the XY scan files from the ScanFiles folder Again once you have copied the scan files they will appear in the iQ 2 Scan 29 Andor Technology Andor iQ USER GUIDE Manager File Edit View Favorites Tools Help sack gt wi jo Search 3 Folders Address Folders E y all Users A Revolution Time Lapse E Application Data O Adobe s LastSession ini E Andor Configuration Settings O Andor Bioimaging I Z 1B C3 Apple C3 Apple Computer O FLEXnet 3 Google C Intelligent Imaging Innovations E Kinetic Imaging a Common Configuration y SnalysisFiles O CalibrationFiles O LUT O ScanFiles z ic mi Dummy O No Grab 9 Revolution Time Lapse E O ios Revolution Time Lapse 2 3 1 5 Setting Up Devices and Hardware If you are using iQ for the first time or on a new PC or system you will need to follow these directions Once the configurations have been set up the Administrator will need to run iQ to set up all the devices used in the configuration Hardware settings for the devices include COM ports board selections and device specific settings such as calibration information filter locations and descriptions etc 30 Andor Technology Andor iQ USER GUIDE 2
262. e been used in testing iQ2 and to establish its functionality within the design parameters 4 6 1 Multi Phase Protocol for FRAPPA FRAPPA protocols require at least 3 distinct phases These are baseline measurement bleach and recovery measurement followed by analysis to measure the T1 2 recovery time which can be used to estimate diffusion rates The Protocol below shows this pattern which can be easily created with the tools illustrated in previous sections Py Protocol Test Case 1 Frappa Multi Phase xa Image Test Case 1 Frappa Multi Phase i Protocol Type Time Series A Camera Binning Use Current Wait Allocation Off Camera Selection Use Current Events dP Pause Click OK to start protocol Move Channel Chi Repeat T 20 times 200 mg al Snap i End TO ar Frappa Frappa PFRAPRA All Move Channel Chi ae 20U times 0 me fastest Se onap i End TO 210 Andor Technology Andor iQ USER GUIDE 4 6 2 Repeating Multi Phase Protocol for FRAPPA Py Protocol Test Case 2 Multi Phase with Repeat se Image Test Case 1 Frappa Multi Phase a Protocol Type Time Series ao Camera Binning Use Current Wait Allocation Off Camera Selection Use Current fogo Events dP Pause Click OK to start protocol gi Repeat T 10 times 5 min JE Repeat T1 5 times O me fastest Ao Move Channel Ch al Snap dy End 11 ar Frappa Frappa FRAPPA All gi Repeat T1
263. e control of 2 lines The 11 screen shot of the Channel Manager above illustrates how to select the Green Yellow source on and off with D7 labeled as Yellow for ease of interpretation Proceed with creating the Channel in normal way selecting the correct settings for the DSD and then on to camera exposure gain LUT etc Note that in TTL mode the LLE will deliver full power by default so your primary control of specimen exposure is exposure time which can be set with high precision in the camera Andor Technology 578 Andor iQ How To Documents 12 Once you have setup the desired Channels you can proceed to build the Protocols you wish An example right shows a time lapse Z series with 3 Channels This appears identical to a Protocol in any other iQ configuration A Came Came QR End Protocol Protocol ia Image Protocol Acquisition Calibration 0 LR_40Xwi ra Binning Use Current R Wait Allocation Off ra Selection Use Current Events Repeat T 10 times 1 min epeat Channel DAPLFITC TRITC Channel Description 4 Repeat Z 10 um in 21 planes sg Snap Frame End Z a End Channel T Protocol Es Protocol Run P Channel iti f Loop Protocol V Display Image F Show All Save As Bi sevens oi Options _ Ore 2 Help Andor Technology
264. e data The data is saved in tab delimited format and can therefore be opened in Spreadsheet packages such as Microsoft Excel 5 4 3 Coincident Statistics The Coincident Statistics Tab opens the Coincident Statistics page Statistics can be calculated here on the user defined regions for all pixels labeled with more than 15 67 14 58 20 46 40 03 21 49 23 04 3 00 4 00 4 00 3 00 4 00 4 00 14 00 14 00 14 00 14 00 14 00 14 00 13 00 13 00 13 00 13 00 13 00 13 00 one color i e RedGreen GreenBlue RedBlue RedGreenBlue 208 Andor Technology Andor iQ USER GUIDE Acquisition Analsis RGE Analysis Calibration Movie Editor Spot Analysis Settings Analyse Analyse Level Statistics Channel Statistics Coincident Statistics Options potes Min Thresh A Min Thresh G Min Thresh B Max Thresh A 4 00 14 00 73 00 1106 00 115 00 3 00 14 00 13 00 106 00 115 00 3 00 14 00 13 00 106 00 115 00 4 00 14 00 13 00 106 00 115 00 3 00 14 00 13 00 106 00 115 00 3 00 14 00 13 00 106 00 115 00 4 00 14 00 13 00 106 00 115 00 4 00 14 00 13 00 106 00 115 00 3 00 14 00 13 00 106 00 115 00 3 00 14 00 13 00 106 00 115 00 4 00 14 00 13 00 106 00 115 00 lf regions have been defined on the current image using the Rectangle Ellipse Polygon and or Freehand Polygon buttons in the Image Toolbar statistics can be calculated on each region for each time frame If no regions have been def
265. e data is plotted may be adjusted using the Chart History spin buttons The user can also insert into a Protocol with a Right Mouse on Snap however this is not applicable for Fast Protocols An example On Line Chart window is illustrated below There are two tabs on the side of the graph One is for the Channel data and the other is for Ratio data Mean Channel Channel Name File Channel Selection Mean Channel Channel Name no PaE Channel Selection Channel See 52 54 56 56 60 62 64 66 66 FO f2 74 76 Fe OU 82 64 86 J2 94 J6 9a 100 205 Andor Technology Andor iQ USER GUIDE On completion of the experiment the charted data can be printed for further inspection and evaluation using the Print option in the File menu Click the Close button to exits from the On Line Chart window 4 5 2 2 Chart Options Chart data presentation depends on settings specified in the Chart Options Channel and Ratio Tabs A discussion of IQ Analysis features in conjunction with the use of regions of interest is described in Section 4 2 Chart Options Channel Ratio Chart Options File Streaming A AMS During Acquisition During Animatior Time C Dimension W Background Correction Group Regions iw Separate Plots wi Suse legen Frame Number f Frame Number Show Event Labels Region Display Chart History fe All Regions iw Enabled 50 C Selected Region Scaling Channel Ratio f Auto Scale Auto
266. e derived equations 19 8 20 in D Axelrod D E Koppel J Schlessinger E Elson Andw W Webb Mobility Measurement by Analysis of Fluorescence Photobleaching Recovery Kinetics Biophysical Journal Volume 16 1055 1069 1976 Note that a fully quantitative calculation is not performed and that a circular beam profile is assumed 339 Andor Technology Andor iQ USER GUIDE ee ANDOR ene Ie CHNOLOGY 6 Processing Tools Andor Technology 340 Andor iQ USER GUIDE 6 1 Process Episodic Average Episodic Average Mask Wizard Field Split Flip Rotate Image Image Filter Rolling Ball Filter Threshold Image ClearView Deconvolution Best Z Image Math Ratio Image Image Image Math Dual Image Disk Episodic Average Image Selection Background Subtraction Photobleach Compensation Image Details 4 Image Disk CAIMAGES DYD V1 4S CALCIUM AFURA TIF CAIMAGES DYD V1 44 CALCIUMSFURA TIFT CAIMAGES DYD V1 44 CALCIUMSFURA TIF The Episodic Average plugin can be found in the Process menu in iQ s Main Window and is used to average multiple repeats of an episode or experiment to produce a resultant image which of the average of all episodes selected The images must be identical in all dimensions e g time XY bit depth wavelengths etc After opening the plugin simply select two or more images from the Image Disk using the standard Windows function of holdin
267. e first well again and move the top edge of the first well of the micro plate under the objective The top edge of the well is the Andor iQ 1 9 1 Andor Camera E File Image List Settings QC Wizards Process Analysis Plug Ins Help Setup Wizard 15 x Camera Orientation Multi Yell Plate and Petri Dish lt lt Back Next gt gt Cancel Camera Orientation 1 Move the stage as shown below so that the TOP edge of the first well enters the field of view Stage Movement Welll 2 Does the Edge appear in image window from OR Bottom Top that mosaics and micro plates are accurately scanned This dialog asks you to make sure you have a feature in your field of view of a small size lt 50 pixel diameter and of high contrast this is often easier in transmitted light but fluorescent beads are also fine even the debris on a cover slip or the previously used micro plate will act as a suitable target See below for an example specimen image nearest edge to the border of the plate Select the Top or Bottom button in the wizard depending on which resembles the Live image in the Image Window Click on the Next button Now follow the steps to ensure that the camera axes are closely parallel to the stage axes This is necessary to ensure Andor iQ 1 9 1 Andor Camera 15 x File Image List Settings Setup Wizard xY Calibration Analysis Plu
268. e for use in Protocol brightfield setup H Channel brightfield Channel Auto Bandit Channel Auto Blue Channel Auto Green Channel Auto Red 847 Channel red auto Press Close to exit the Channel Manager 531 Andor Technology Andor iQ How To Documents A5 2 How to Set Up a Multi field Scan 1 Select Scan from the Acquisition Tab or the File Menu to open the scan Manager 2 Select New to create a new scan Enter a name for the new scan 3 Select Multi field Scan option 4 Check centre on Registration Point if you need to use a reference point on the slide well Press Next to continue Channel Names Select channel to go live with for Scan setup bright field ooo 37 a gt Setup Wizard Scan Set Up New Scan Next gt gt Scan Setup TFG DO y i scan Options M Multi Well Petri Scan M Multi field Scan Centre on Registration Point Zseries Please enter a name forthe new Scan Carrie s field scan 5 Select Channel to go live with for Scan setup Press Next to continue 6 Type the desired Number of Fields into the text box mMulti_Field options Select Number of Fields ai Press NEXT FIELD to select position and mowe to next field Press PREVIOUS FIELD to select position and move to previous field Please ensure im
269. e g at 2x2 binning exposure will be multiplied by 1 2x2 0 25 Return to Table Camera Selection Edit this item with Current or if there is more than one camera loaded into iQ with the camera D specified in the Camera Selection page of the Device Setup Dialog In this way a Protocol can be associated with a specific camera and you can switch cameras using the Load Protocol Action Return to Table Delay Set a delay period ms seconds minutes hours between Actions This Action can be used to ensure that a state change e g Channel or XY has reached Stability before proceeding Return to Table 195 Andor Technology Andor iQ USER GUIDE End Always inserted with a Repeat Action and therefore associated with Montage T Well XY or Z e g End T1 or End XY If there is nesting of Repeat T loops then each loop parameter has a postfix single digit T1 T2 etc and the associated End will have a matching index e g End T1 End T2 IMPORTANT NOTE When you insert any Repeat Action an associated End Action will be included immediately afterwards You can drag this End Action downwards to enclose following Actions as you wish e g Channels Snaps etc Return to Table Frappa execute a Frappa Action specifying the device FRAPPA MicroPoint or Mosaic Frappa Channel and Single or All regions The last parameter defines whether the currently selected Frappa region will be stimulated or all Frappa region
270. e image Click the mouse button at the position of the first vertex of the polygon and then drag the mouse on the image to define the rest of the polygon Right click to end the construction of the polygon Freehand Polygon regions can be moved and deleted with the Pointer or Hand 98 Andor Technology Andor iQ USER GUIDE button but cannot be resized Right clicking on the image or a selected ROI will also bring up the same menus as discussed in Section 2 10 1 2 10 13 The Define Text Button AET Region Type Text T j EN The Define Text Button will allow aw E OE the user to insert text onto an Width Height 233 so image After selecting the Define Text button use the left mouse button to define the upper left hand of a box and drag mouse until box is the necessary Em Insert text by right clicking test bos and selecting Edit from size Release mouse button Right click on box and the ist Acuust Region Type color font and message as necessary Click ok to accept select Edit The Region Details dialogue box will appear allowing the user to change the size of the box select a ROI Classification change the font and enter a message Select the Ok button to continue 2 10 14 The Information Field Button e Selecting the Define Stamp Field Button brings up the menu seen T i mo i do on the right Select the item from the list to insert and use the right mouse button to drag a box on the im
271. e is selected Add Chart Options Channel Ratio Chart Options IV Background Correction Axis H Group Regions During Acquisition During Animation ose WV Separate Plots C Time Dimension Show Legend Frame Number Frame Number Show Event Labels baa Background Colour E M Chart History Region Display O Enabled 50 EH Save All Regions Bes A Speed Regen rScaling Channel Ratio Auto Scale Auto Scale Manual Min fo C Manual Min fo Max 256 Max 3 540 Andor Technology Andor iQ How To Documents A5 8 How to Open an Image TIFF 1 Select Open Image from the File menu in either the iQ control window or the image window Andor iQ Amta AE Image List Settings QC Wizards Analysis Process Image Edit Plug Ins Help Open Image Save Image Becca ee dministrator l e ANDOR O Settings Orca ER Ludl Piezo AT BMP Exit Image Importer ImageType 8 bit grey pseudo 146 Image Size 320 x 240 Humber of Planes 1 Display Range 1 to 146 eee cet sas 2 This will open the File Browser window Select the drive and folder in which the images are located By default iQ will look for TIFF files If you wish to open images of a different type use the dropdown menu Files of type Open File My Recent Documents O Te Desktop ip EES My Documents Documents x My Computer a 3 Floppy A
272. e left hand edge of the well is the nearest edge to the border of the plate Select the Left or Right button in the lt lt Back Next gt gt Cancel wizard depending on which resembles Camera Orientation 1 M h h bel hat th i i i LEFT edge of the first wall antars the fiald of view most strongly the Live image in the Image Window as the stage plate makes its way gt to your right weli y At this point you click on the Right button 2 Does the Edge appear in image window from i Let oR Right if you see the left edge of the first well move into the field of view from the right and Left accordingly If you choose Right you may see the message below and you may have to rotate the camera physically by 180 degrees to ensure correct scan operations If you can not rotate the camera you can modify the camera orientation in the device setup dialog if your camera supports this feature see the beginning of this chapter for more details Orientation Warning Please rotate the camera through 180 degrees AN or select rotation option on camera options panel if available To access camera options qo to device setup and select camera Then click on OK and IQ will return to the stage above where you will need to rediscover the left edge of the first well 127 Andor Technology Andor iQ USER GUIDE Now relocate the approximate center of th
273. e manual mapping icon and adjusting the parameters as described previously in Section 5 2 In addition the Automap button at the bottom of the Image Window can be used You will need to click on the Orthogonal Slice View Window to update the view with the newly selected mapping parameters Clicking on the Options button opens the Render Options panel shown below 300 Andor Technology Andor iQ USER GUIDE Orthogonal View Options Depth Dim z 29 Include Intensities Between ooo 1 00 Anim Dim Time A F Display Mode 65535 00 Single Slice CO Maxinum Intensity Zoom Factor CO Minimum Intensity ay 1 7 E i 7 Best Slice Depth Dim you can choose which dimension to use as the third dimension for the orthogonal rendering Anim Dim you can choose which dimension to use as the animation dimension Display Mode gives you several choices such as Single Slice Maximum Intensity and Minimum Intensity Render Methods Single Slice gives a map of the points that appear at the position selected by the crosshairs while Maximum Intensity and Minimum Intensity give a two dimensional projection of the maximum or minimum intensities along the lines of view through the volume Include Intensities Between this has two scrollbars that you can use to select the range of intensities for rendering Zoom Factor this allows you to change the zoom along all XY and Z axis Best Z Slice checking it w
274. e name Files of type decon testi2 tif Ei decon testis tif E decon btesti4 tif E decon testl5 tif E decon testiG6 tif El decon testi tif E decon testlis tif E decon testla tif E decon testzo tif El decon testzl tif E decon _testzz tif JPEG po pea Kontron Micro Vowel MIF Multi Load Photometricos Princeton Instruments Targa image will be opened decon_testz3 tif E decon _testz4 tif E decon testz5 tif E decon testz tif E decon teste tif E decon _testz8 tif E decon best ti E decon bests tif E decon best31 tiF E decon_best32 ti E decon testas ti O Andor Technology Open File 1 Olympus GE 200 hi Olympus GE200 Image Cytometry Standard ics ah decon best34 tif decon_best35 ti E decon _test36 tif E decon bests tif E decon testas ti decon test3a tif E decon _test40 ti E decon btest4 1 tif E decon best42 tif decon _test43 tif E decon_best44 tif Open Image open an image from disk If you open an a image while Live the system will return to Idle and the The example below shows the dialogue when opening Multi Load enables the import of multi 2 A Cancel Andor iQ USER GUIDE Open File Look in Cathy2_confocal_20100722_135321 d E My Recent Documents Desktop My Network Places Once a file has been selected and the Open button pressed the status of the import operation is dis
275. e palettes are selected the default palette is used At the bottom of the LUT list there are three options available Load Edit and Save The user can load a previously defined LUT by selecting the Load option This will 239 Andor Technology Andor iQ USER GUIDE open up an Open File Dialog and the user can select the LUT files they want When Edit is selected an Edit LUT Dialog is displayed from which the user can modify the current LUT by adjusting the graphic presentations of its Red Green and Blue components Edit LUT for Multi Load xj Red 0 Grey Level 255 bo vi 154 168 Help DISF RED GREEN BLUE The modified LUT can be saved as a new one if Save is selected later The LUT pop up list will show add user defined LUTs to its List once they have been saved Another way of creating new LUTs is from a text file based exactly on the format of the LUT file The LUT file format is illustrated over 240 Andor Technology Andor iQ USER GUIDE FORMAT of LUT FILE Description ASCII LUT File TRUE Red Green Blue 000 000 000 000 001 000 001 000 002 000 002 000 003 000 003 000 004 000 004 000 005 000 005 000 006 000 006 000 007 000 007 000 008 000 008 000 009 000 009 000 250 000 250 000 291 000 251 000 252 000 252 000 253 000 253 000 254 000 254 000 255 000 255 000 You can create this in Notepad or you favorite editor and then save the file to the LUT folder which can be found here
276. e to set the address A two position jumper selects a range from 1 10 or 11 20 and then a ten position jumper selects the actual address 480 Andor Technology Andor iQ USER GUIDE For instance if the selected range is 1 10 and position 7 on the ten position jumper is selected the module is set for address 7 Whereas if the selected range is 11 20 the same ten position ADDRESS 1 ADDRESS 2 JPI JPI oo 19 o jumper setting would 288 ob 2066 306 represent address 17 A 100 100 seo seo module can be set to any 600 see 706 700 seo see address in the allowed ane alae IUe o iue e range The high level ii ina J J interface functions ADI 10 OS ADII 20 ADI 10 ee ADI1 20 however assume that ADDRESS 11 ADDRESS 17 i JPI JP1 certain modules of a Os we 200 200 certain type are set for soa aos S EE iee specific addresses See 50e 500 see 6ee Charts of the DIL zoo 7 oo seco seco cee xes connectors right o moa pa wees JP5 JP5 AD1 10 ee ADI1 20 AD1 10 ee ADI1 20 481 Andor Technology Andor iQ USER GUIDE A3 7 ASI XYZ Stages A3 7 1 The ASI Settings Tab There are four sections included in the ASI Settings Tab Camera ASI Sutter Position Hardware xXY Stage z Settings Communication Position Establishing Communication Hardware 7 Reset V Stage Control IV 2 moter control Load Position is located at the top of the Settings Tab This will show the L i n ME icati c
277. e wavelengths where microscope optics are well corrected and transmit strongly the selection of optical components is not too critical though alignment and focus must be handled with care But at e whole cells organelles individual filaments such as actin and microtubules and at even lower energy levels the system can be used for photo bleaching need to know what objectives you are using details of mag NA and Nikon model number This will allow us to establish the UV transmission properties of the lenses The fact that you can induce specimen damage proves there is enough light coming through the system For uncaging focus is critical because it is typically done at energy levels below bleaching Using the first surface mirror for setup is only a first step because you must account for the 564 Andor Technology 965 Andor iQ How To Documents cover slip and any medium in which the specimen exists One of our experienced engineers from PI has suggested a 3 or 4 step focus process 1 Take a slide in which you can identify single microtubules or actin filaments or similar and with a DAPI cube in place and mercury lamp illuminate the specimen the UV cube should be transmitting 365 400 nm mercury has a 365 and 405 nm line Get an idea of how long the filaments take to bleach to 50 intensity a camera helps This time will give you an idea of how long you may need to pulse the 365 nm MicroPoint for similar effect
278. eat Channel Repeat Montage Repeat Time Repeat Well Repeat XY Repeat Z Snap Frame Snap Stream Trigger In Trigger Out Wait Allocation XY Scan 194 As In fo fo Q e 7 Aa Set ScanZ stage to specified location Show region of interest intensities in chart Show user defined message and wait for OK for internal use only Compute on line Ratio lterate through list of Channel lterate through overlapping fields for mosaic lterate n times with defined interval t lterate a series of Wells in a multi well plate lterate through XY coordinate list lterate a series of Z positions Z scan Acquire a frame Acquire a frame in Stream mode device sync not guaranteed Overlap ON OFF Wait for specified Trigger input pattern Output specified Trigger output pattern Wait for image allocation to complete before lterate through XY coordinate list Andor Technology Move Scan Z On Line Chart Pause Ratio Repeat Chanell Repeat Montage Repeat Time Repeat Wel Repeat XY Repeat Z snap Snap Stream Trigger In T
279. ector and 32 Mbytes of free hard disk space A3 2 1 2 Device Setup The camera setup is accessed from one of the Device Setup buttons located on each window of iQ Device Setup Each piece of hardware in the selected profile setup in the Configuration Manager will have a tab labeled accordingly In addition to the tabs the following Options are available at the top and bottom of each Tab Current Channel is located at the top of each tab under Device Setup This allows the user to go live or snap an image with a channel by selecting from the dropdown menu and the record to changes to the channel settings Acquisition Auxiliary Devices test M n Live Camera Prior 447 Andor Technology Andor iQ USER GUIDE Command Status is located at the bottom of each tab This will signify the cameras or devices operating status and allow the user to reset the camera at any time The user can also press the Andor Icon under the reset button to display the About Box About fx Andor Bioimading formerly Kinetic Imaging Product Kinetic Dummy Camera Acquisition D evice Version 5 0 0 733 BuildTime 13 19 17 Feb 7 2005 Copyright Andor Bioimaging 2001 2005 Command Status emess O Camer Camera is woking III working i Strelley Hall 100 Southcenter Court Strelley Suite 9006 Nottingham Morrisville NGS GFE UE NC 27560 USA tel 44 115 906 1354 tel 1919 467 O60 fax 44 115 906 1
280. ed The system can be left to self start after Allocation If you leave the system to self start make sure there are no Pause Actions following Wait Allocation Return to Table XY Scan shows the XY Scan name currently in the Tree When you insert an XY scan in the Tree it creates a Repeat XY iterator and if the XY Scan includes Montage and Well settings also a Repeat Montage and Repeat Well iterator Note that Autofocus can be used on all these settings provided the Autofocus device has been initialized when Protocol execution begins Return to Table AutoSave is controlled by the checkbox and path setting shown below which is resident on iQ s Acquisition page If the box is checked iQ saves the Protocol image data when the Protocol ends execution Images are saved in multi page TIFF files with the root name specified in the Image Action iQ will add date and time postfixes to the filename The folder name where the data will be saved can be set by clicking on ellipsis button Return to Table Auto Save E C MAndor iQD ata M Browne Revolution Time Lapse4utol mages 202 Andor Technology Andor iQ USER GUIDE 4 5 Protocols basics In this section we look at how to create and edit Protocols for some specific scenarios 4 5 2 Creating a Two Channel Time Series Protocol The following example describes a protocol used for dual excitation ratio imaging measurements using the Calcium ion indicat
281. ed allowing and map bleach positions M Manual Position Selection the calibration to flow more easily Now with the specimen in focus select a suitable Frap channel and click the Bleach Sample button If the system has been pre aligned you should see in the image a region of the specimen which has been bleached A bleached region will appear darker than the rest of the specimen and should be localized Note if the bleach cannot be seen you may need to scroll the XY stage a little or increase the FRAPPA time if the bleach is still not visible This step of finding the initial bleach may be tricky the first time you calibrate the system Once the bleach spot has been identified in the field of view then you must create corresponding pixel coordinates and angles for the calibration procedure To do this for each bleach point region click on your best estimate of the region center with 142 Andor Technology Andor iQ USER GUIDE the marker and then click on the button labeled Add Calibration Point e FRAPPA Calibration View Setup Wizard FRAPPA Calibration sani BR WIAA Niele DOlO S T i mi eo E set up FRAPPA alignment and calibration settings lt lt Back Next gt gt Auto Calibration Point 0 658 570 128 126 Point 1 714 637 153 151 Point 2 595 639 154 102 Saint Selection Add Calibration Point 3 103 0 105 0 Delete Last Point Bleach Time and Channel
282. ed delay settings Transition delay is the time iQ allows for a shutter to open or close e g wait before acquire frame In general the LLE is so fast you can set Transition Delay O ms Note that because the Green Yellow channels use a mechanical switch to select wavelengths that a Transition Delay 100 ms setting should be used for output 7 see paragraph 6 Test your device below This will ensure proper synchronisation between LLE illumination and camera exposure Other output Transition Delays can be left at their default values of zero Interval is the duration of a pulse which is used with High Low or Low High and defines the interval for which the TTL level is asserted High or Low respectively Pulses can be used for various functions e g synchronization Andor Technology Andor iQ How To Documents 4 The LLE product can be sourced in various configurations with 3 to 7 wavelengths The Table right shows how Wavelengths colors map to Generic Shutter digital lines DO D6 The mapping is invariant between models i e Red is always controlled by DO in the LPT port and so on Note that Yellow and Green share a source and D1 must Green Yellow Cyan D2 Cyan Blue D6 Reserved _ D7 G Y Low gt Green High gt Yellow be set in conjunction with D7 to switch Green or Yellow On 5 Rename the rest of the Shutter outputs as necessary depending on your LLE configuration 6 Test your device make sure Ou
283. edit TOREN ATIRAR Readout ms 32 034 box Actual Frame Rate 0 00 Note Camera exposure times will automatically adjust when binning is changed The Actual Exposure Time displays the current exposure time The Readout Time is the exposure time plus the time it takes to get the picture from the camera to the computer This gives an indication of the maximum possible frame rate Experiment Control or Trigger Control Expernment Control Fast Lambda ed Foeni foo o f Free Aun Ext Trigger Trigger Contro ms f FreeRun C Ext Trigger Timeout 1000 Fast Lambda Zed Automatically synchronizes the camera with a piezo focus drive and Sutter DG4 Monochromator or AOTF controller when capturing at fast speeds This option is supplied along with special purpose hardware as part of the Andor iQ FastLZ module and uses a hardware driven External Trigger to the camera piezo and wavelength 152 O Andor Technology Andor iQ USER GUIDE controller Free Run Mode This is the normal mode of camera operation exposure will be initiated from within the software without the need for any external triggering External Trigger Mode This option uses an external device to initiate the exposure The Ext Trigger input on the camera body needs a TTL signal input to ensure exposure is started Exposure begins on the positive going edge of the signal and completes based on the software exposure time setting Time
284. edonion Z Protocol tesing a Image Default i Protocol Type MU Camera Binning 1 1 Events Pause Click OF to start protocol eB Interval 500 ms Repeat 10 98 ms Fastest Channe a Snap Edit Channe Cancel N Snap End 3 The right mouse click brings up the Menu on the right Please note that acquisition will PAUSE during this process 4 The first option is 88 ms fastest This is the fastest possible speed the protocol can run with the existing hardware configuration Edit interval length ms F Cancel 5 The Second option is Edit This brings up the Edit interval length Type in what you would like to change the interval to and select the unit of measure from the drop down list 536 Andor Technology Andor iQ How To Documents 6 Select Cancel to exit and allow Acquisition to continue 7 The new Interval Setting will now appear in the Protocol Tree and Acquisition will resume Protocol teasing m Image Default i Protocol Type ME Camera Binning 1 1 E vents Pause Click OF to start protocol en Interval 1000 me es Repeat 100 times 4 Channel Blue 1 Snap Channel Green 2 Ew Snap End 537 Andor Technology Andor iQ How To Documents A5 6 How to Use Load a Protocol Protocol tesing 1 Select the Protocol Make sure the Image Default f Protocol Type MC Camera Binning
285. egions on the image The results are displayed both graphically and in numerical form Acquisition Analysis AGE Analysis Calibration Movie Editor Spot Analysis Analyse Save Bl Graph Data Options 5 3 1 The Graph Page Acquisition Analysis RGB Analysis Calibration Movie Editor Spot Analysis Analyse Save Settings Graph Data Options Mean J Auto Range Show Event Labels Show Legend Print BA Mean Intensity we Mean Intensity 810 800 790 780 770 760 750 Settings lf regions have been defined on the current image using the Rectangle Ellipse Polygon and or Freehand Polygon buttons in the Image Toolbar Statistics can be calculated on each region for each time frame The statistics to be calculated are selected on the Options page The calculated plots are drawn on the Graph page while the calculated statistics are displayed on the Data page Pressing the Analyse button will perform the analysis Note If no Regions of Interest have been defined the analysis is calculated for the entire image 246 Andor Technology Andor iQ USER GUIDE Once an analysis has been performed pressing the Save button will open a save dialogue window allowing the user to specify a directory and file name to which to save the data Note If Automatic Directory Selection is selected on the Options page the default folder to which the data
286. elete Reset Ok Cancel Help The Dimension Editor is used to change dimension names and units or to remove redundant dimensions from an image that is currently displayed in the Image Window 75 Andor Technology Andor iQ USER GUIDE Dimension Details A Dimension editor has a special function for apenas 5 Channels as shown left to edit Channel AO faiava stamps and names This can be useful in m aeee cases where wavelength stamps have been mooo assigned the same value and then analysis cannot proceed Changing the Stamps to Units Resolution Origin 0 different values will correct the problem Edit Channels Name and Value Current After using the Multi Load file opening tool which defaults the 3rd dimension to time you may wish to edit this for visualization or analysis purposes to Z wavelength etc A dimension is removed from the table by pressing the Delete button the deletion process works sequentially such that the highest dimension in the list is removed first For the example shown above pressing the Delete button will remove the 8 and 9 dimensions from the list The Dimension Editor panel following the deletion of all redundant information is shown below 76 Andor Technology Andor iQ USER GUIDE Dimension Editor M af Name MFMONZT2CH 2x2 16102009 TIF Type 14 bit grey pseudo Intensity 1 000000 lux x 502 0 400000 um x Dimension Dimension Name Resoluti
287. ely between the 2 images for reasons that will become clear later Adjust to match angle and XY Using the Align Camera Plug In which is accessed via the Wizards menu select the 2 feature method to allow you to measure orientation and XY position of 2 user selected spot The camera physical rotation should not need further adjustment but the cassette does allow for some adjustment of angle of the beam splitter angle Use the DPC cassette adjustments see the description at the end of this document and figure below to optimize the XY positions of the spots until the Plug In reports angle X Y positions match as closely as possible Note that the dichroic mirror in the DPC cube introduces a vertical flip of the image from that camera so make sure to introduce the correct flip rotate in 120 Andor Technology Andor iQ USER GUIDE the Camera interface prior to matching alignment If this is done correctly you will be able to match angles between the two cameras within a degree or so when well aligned Note also that angle or rotation is most critical here as small translation errors can be adjusted in the next phase using the alignment software Once this step is done reset the Flip Rotate option on the camera GUI to None A maximum of two regions can be used for the alignment procedure Select 2 Regions for registration by clicking on the appropriate radio button 4 Lock down and adjust cassette Once you have comple
288. ement of background data acquisition A3 2 2 2 2 2 The Speed Amplifier Tab Exposure General Speed to Readout the signal from the camera _T meetstue Shuiter_Speed Ampliir Image Orientation Readout Speed sensor a combination of vertical shifts and Horizontal Readout Time Vertical Readout time 10 000 MHz 1 000 us horizontal pixels clocking is required This dialog Use Defaut ADC Channel Vertical Clock Voltage lets you set the vertical shift time Ty and oo x mm Horizontal Frequency Fp values 3 MOutput Amplifier f EM Pot Conventional j Baseline Clamp Horizontal Vertical Readout Time In the course of readout charge is moved vertically into the shift register and then horizontally from the shift register into the output node of the amplifier Therefore the user can specify both the Horizontal Readout Time and the Vertical Readout Time 455 Andor Technology Andor iQ USER GUIDE The faster the readout speed the more readout noise is created however the electron multiplying structure can amplify the low signals above the readout noise The default values are shown in the picture above The Readout time for a sensor of H by V pixels is computed as follows Readout Time V Ty H Fx ADC Channel Analogue to Digital Converter Channel can be selected depending on the readout mode for the DV887B cameras When these cameras are operated in conventional readout mode no
289. en labeled On and Off for clarity Once you have set these up as desired click on the Lock button to fix the settings When the Insert Trigger IN dialogs show next time the lines will show their newly labeled states both in the Protocol Tree and the dialogs If you change labels in the Trigger Setup panel you must revise Edit the Trigger entries in the Protocol Tree since they will not be automatically updated and will fail to be Acquisition Auxillary Devices E x matched with the original entries Camera Trigger Input Output Set Lp Bul Ff FF F FP of The Trigger OUT setup is accessed in the Trigger Name Picospritzer III same way and edited similarly In this A example the signal will be used to m activate a Picospritzer injection device DU S which will apply a pulse of agonist to the U kow Hin loin IO cells under study _ Output Line 0 has been selected and the line state HIGH LOW has been renamed to Pulse This will deliver a pulse as shown the duration is set by typing a value into the interval box this value is the pulse duration in ms 231 O Andor Technology Andor iQ USER GUIDE 4 8 3 Using Events and Markers The Events command is inserted into an IQ protocol by default To adjust the way the Events will be used you simply Right mouse on the Event command in the Protocol and Edit Events will create markers in the Image at the time point when they occur and these markers will be availa
290. ence region and disabling correction from the Pre bleach Correction menu Pre bleach correction As above but enable the correction from the menu Reference region correction Select an ROI in step 4 above and enable the correction from the menu To view the added region select the created region and click Update Point Data Table 5 13 4 Defining and Modifying FRAP Periods and Intervals In order to carry out the calculations the user needs to define where the four FRAP intervals occur e lf the user selected All in any of the Point Position checkboxes then there will be several rows populated in the Point Data table Choose the one that is of interest The chosen curve will become highlighted in the chart e Right click on the chart and select Insert Period e Move each of the five red lines so that the four intervals are properly defined The first four lines define the point at which an interval starts This is illustrated below The ellipses contain the points included in each of the distinct intervals Andor Technology Andor iQ USER GUIDE Prebleach leac Recovery Sates ate The end of the Steady State interval sets the extrapolation extent i e the curve is fitted to the data in the recovery region and this curve is then extrapolated out to the end of the Steady State interval The calculated values are derived from the extrapolated curve Note that the extrapolated curve is only visible when plotting in real time
291. er of the Image Window When unchecked the image origin is located at the top left of the Image Window 83 Andor Technology Andor iQ USER GUIDE Chart Image formerly On line Graph opens an intensity real time plotting window for the whole image or a user defined area Sections 4 4 2 1 and 5 2 Strip View opens the Strip View dialog where you can create a file strip type view which is useful for showing changing processes or Z planes on the printed page or when movie capability is not available 4D View opens up the 4D View Window to display a high quality volume rendered view of the current data 4D View shares much functionality with Fast MIP except that it renders the data set with higher quality at oblique angles 3D View opens up the 3D View Window to display a three dimensional view of the current image Section 5 3 Montage View opens up the Montage Window and allows you to stitch images together into a large field of view after they have been acquired in montage or mosaic scan modes Montage is sometime called mosaic or tiling images Kymography Allows oblique and polyline slicing of time series and other multidimensional images Section 5 4 Orthogonal Slice View opens up the Orthogonal View Window to display orthogonal slice views of the current image Section 5 5 84 Andor Technology Andor iQ USER GUIDE Clicking on the Toolbars option displays the dialogue shown below
292. er window from which the directory containing the images can be selected In this example the beginning of the file name FURA time is common to all the files we wish to import so this is entered into the Common File Name box Pressing Next will show the following step In this step the dimensions of the images must be entered In this case there are two channels and series through time lf there is only one channel untick the Multi Channels checkbox Press Next 45 Andor Technology Andor iQ USER GUIDE Image Importer Wizard i Now the two channels must be identified by their file names Image Import Options Channel 0 files end wave0000 and Channel Set Channels 1 files end wave0001 These are entered in the Channel Identifier boxes respectively pora f and the wavelengths if known in the Wavelength nm boxes Channel Identifiers should be a text string that it contained solely by the file names for a channel If there is more than one channel chosen each Channel Identifier should be different from the others Press Next Channel Identifier Wavelength nm 0 waveD000 340 1 wave000 390 Files JFL RA timeQO00_ wave000 tit d xi Whereas the wavelength identifier was wave0000 and wave0001 the time identifier Image Import Options in this example is just time The units are eae known to be approximately 0 6 seconds starting at time 0 Specifying
293. erneath the scroll bar Cooling the CCD detector helps you reduce dark signal and its associated shot noise Also the impact ionization that yields gain has negative temperature dependence increasing by a factor of 10 as the temperature drops from 0 C to 50 C The camera will commonly be run at 70 C for low light microscopy when the gain can exceed 3000 or 4000 Although the camera is not damaged by high levels of light exposure we recommend that the gain be kept low when not in use to avoid excessive camera saturation and potential aging of the EM gain which may need to be factory reset after thousands of excess saturation exposures 454 Andor Technology Andor iQ USER GUIDE Camera Fan Control Allows user to specify Full soeed low speed or off by selecting the corresponding radial button The camera can be run only for short periods without the fan Shutter Under settings the Check box for TTL Transistor Transistor Logic High Open signifies that the system will cause the output voltage from the controller card to go high to open the shutter The Open Close Transfer time is the specified time the shutter will take to open or close This time is used to give the shutter enough time to close before background data are acquired The system ensures that a period of at least the duration of the Transfer Time elapses between the shutter output signal entering the closed state and commenc
294. es a zero stage position If the zero stage position has previously been set click on the Cancel button to continue The Prior pages can be accessed by pressing the Device Setup button on the Acquisition page of your Andor software and clicking on the Optiscan tab These display the current settings of any active devices and also enable the stage to be zeroed without the necessity of exiting the iQ software and starting the session again 470 Andor Technology Andor iQ USER GUIDE A3 5 2 Optiscan Tab A3 5 2 1 XY Stage Clicking on the XY Stage tab Acquisition Auxillary Devices x Camera ptiscan displays the panel shown on the left and enables the Device xy Stage Z Settings Resolution to be specified for the X and Y axes Device Resolution microns step 1 0000 Absolute Position fn OO00 microns At the bottom of the panel the current X Y Z position of the stage is displayed For the example shown the X Y Z stage coordinates are 8251 3199 and O respectively Device Resolution micransstep 1 0000 Absolute Position fn ooo micron 8251 Y 3199 70 l Enable Joystick To allow the stage position to be Receive 8251 3199 adjusted manually using a joystick the Enable Joystick checkbox should be ticked Commands being written to and received from the COM Port of the PC are also displayed at the bottom of the dialogue Communication may be monitored b
295. es specified in the Multi Wavelength dialogue on the Grab page e g for each of the red green and blue planes of a RGB image Acquisition Analysis RGB Analysis Calibration Movie Editar Spot Analysis Settings Grey Level Statistics Channel Statistics Coincident Statistics Options Notes Image ean C DEMOIMAGESSKOMET IMAGESSKOMET 03 TIF 55 66 2 CADEMOIMAGES KOMET IMAGESSKOMET 03 IF 1 71 87 3 ICADEMOIMAGES KOMET IMAGES KOMET 03TIF 2 49 46 4 CADEMOIMAGES KOMET IMAGES KOMET 037IF 3 71 59 O 5 CADEMOIMAGES KOMET IMAGESSKOMET 03 7IF 4 900 6 CADEMOIMAGES KOMET IMAGESSKOMET 03 IF 5 6016 7 CADEMOIMAGES KOMET IMAGESSKOMET 03 TIF 6 8587 B ICADEMOIMAGES KOMET IMAGES KOMET 03 IF 7 65 34 9 CADEMOIMAGES KOMET IMAGESSKOMET 03 7IF 8 67 63 O 10 JCADEMOIMAGES KOMET IMAGESSKOMET 03 IF 9 65 95 11 JCADEMOIMAGES KOMET IMAGESSKOMET 03 IF 10 61 53 lf regions have been defined on the current image using the Rectangle Ellipse Polygon and or Freehand Polygon buttons in the Image Toolbar statistics can be calculated on each region for each time frame If no regions have been defined Statistics are calculated for the entire image The calculated statistics are displayed in the chart The Settings button opens a dialogue in which you can specify what region of color space to consider Note In order to access the Settings button the Threshold option must
296. essful registration of you devices check the box below Show REGSVR32 EXE message Box 434 Andor Technology Andor iQ USER GUIDE The Andor Configuration Wizard can be exited at any time ignoring any changes by clicking on the Cancel button Once the appropriate camera and auxiliary devices have been chosen click on the Finish button to complete the device activation procedure The Show REGSVR32 EXE checkbox at the bottom of the dialogue IS enabled by default When the i DilRegisterServer in KIDAndorA TA ocx succeeded Finish button is pressed the devices will be registered and a series of message boxes displayed indicating whether the devices have been successfully registered These messages can be inactivated by disabling the Show REGSVR32 EXE checkbox We recommend leaving the box checked to make sure all devices register correctly 435 Andor Technology Andor iQ USER GUIDE A2 1 4 Editing an Existing Configuration t Device Configuration Manager File l Settings Tools About Exit Edit Configuration Delete Configuration New Edit Delete Current Configurations i ie Select the appropriate configuration file name from the Current Configurations list displayed in the Configuration Wizard please refer to Section A2 2 for further details The chosen configuration can be modified by pressing the Edit button or selecting the Edit Configuration item from the Edit menu A Wizard d
297. ested and sequential Z scan loops place holders for Frappa events and online processing Andor provides several templates with the iQ installation but the more advanced user may wish to generate their own An iQ protocol is defined by several files Protocol Channel Ratio and Camera settings A template is basically a Protocol file that is located in a special location By default when a protocol is created the Channel Ratio and Camera settings are added using the current state the user is responsible for modifying these as required 566 Andor Technology Andor iQ How To Documents The steps for adding a template are 1 Create a protocol and give it a name that will represent the acquisition pattern i e My Template 3 Andor iQ 2 2 Andor Camera 4 Ioj xi File Image List Settings Wizards Process Analysis Plug Ins Help Setup Wizard FF vo Protocols Protocol Manager Please Enter Name for Protocol MyTemplate Select Cancel Protocol Templates Current Protocol Time Lapse mi Protocol Time Lapse H Protocol MC 7 Protocol MCZ 2 First 7 Protocol MCZ 1 4 Protocol MultiPhase 1 4 Protocol MultiPhaseWithFrappa Protocol MultiRate 1 4 Protocol Ratio 2 Build a protocol with the basic pattern that you require i e Three sequential time loops with a frappa event in between eachrepeat loop one frapping the nuclear regions the other frapping the c
298. ey are actually interlaced in time when they are displayed The eye cannot detect this effect but with digital processing and analysis it can be a problem This function allows each interlaced frame to be separated into two fields with half vertical resolution but twice the temporal resolution 373 Andor Technology Andor iQ USER GUIDE 6 10 1 2 Operation Source Image Running UMT Select Select an image to use This will default to the image currently in the Image Window Press the Select button to choose a different image This will open the Image Selection Image Details Name Default2 Type 12 bit grey pseudo Intensity 1 000000 lux x 320 1 100000 pm y 267 1 120000 pm Wavelength 2 Time 100 Approximate save size User C Lowe 34 2 MB Configuration Dummy Camera Created Date 21 02 2005 Time 3 20 35 PM Window Height 267 Binning 1 Image Width 320 Image Height 267 Exposure 40 Protocol Description Channel Image Default Camera Binning 1 1 Events ents Image List Image Name Type ESI Frames Z DEVELOPMENT TEST 12 bit gr 102 Default3 12 bit ar Z DEVELOPMENT TEST 12 bit gr Z DEVELOPMENT TEST 12 bit gr Z DEVELOPMENT TEST 12 bit gr Z DEVELOPMENT TEST 12 bit gr Z DEVELOPMENT TEST 12 bit gr Z DEVELOPMENT TEST 12 bit gr Default2 12 bit gr Default1 12 bit gr Image Selection Window Highlight an image fr
299. f each filter are used to specify a minimum and maximum value for that parameter Values outside the specified range will be ignored To alter a value type the new value into the text box After adjusting the filter range or selecting deselecting a filter clicking on the Filter button updates the filtered image window This image can be compared and the effects of individual filters or combinations of filters optimized 290 Andor Technology Andor iQ USER GUIDE ee P Spot Analyst Aa sts so md CATEMP SPOTFILTEREXAMPLE TIF_Filtered No filter applied P Multi SaaS eae J E pot Analysi is j CATEMP SPOTFILTEREXAMPLE TIF_Filtered Filter combination applied Filter Min Max Area 500 1055 Shape 0 39 1 P Multi Andor Technology 283 Andor iQ USER GUIDE The Reset Default Values button restores the Min and Max parameters to their default values HINT The Min and Max range settings provide limits for feature detection If you are setting up you may wish to change these to include a wider range to ensure the measurement of all objects that are likely to occur rather than just the range analyzed in the set up Any changes to the proposed protocol and their subsequent effect on the current image can be evaluated by moving through the set up wizard using the Back and Next buttons changing the threshold and filter values where appropriate Note Please note that Spot Analysis disca
300. f operation Set Frame specify the parameters for each of the images in the List as described below Play List after setting up the parameters play the animation before saving as an AVI Movie 269 Andor Technology Andor iQ USER GUIDE In Set Frame mode you can specify the following parameters for the selected mage Starting and Final Frame Using the animation buttons or scrollbar animate to the first frame to include and press In then to the final frame and press Out The frame numbers will be updated in the In and Out columns in the table Note The numbers of the first and final frames of the image are displayed in the Min and Max text boxes The current frame is displayed in the Frame text box Speed of Animation Rate specifies the number of frames to display every second Type a value into the text box or using the scrolling arrows the value will be updated in the fps column Number of Repeats Enter the number of times you want the image to play during the animation type a value or use the scrolling arrows Animation Dimension For multidimensional images that contain more than 3 dimensions select the dimension along which you want to animate 270 Andor Technology Andor iQ USER GUIDE Movie Scaling Factor The sampling factor for the image when saved as an AVI Movie A sampling factor of 0 5 will result in a half sized image 0 33 a third sized image etc Note that you should NOT Zoom t
301. feature for rejection A feature can be rejected from the data analysis by clicking on the appropriate check box using the left mouse button the check box subsequently appears blank Data for all the features analyzed is selected automatically by default A visual appreciation of the data rejected from the analysis can be gained by using the Show Data function in combination with the Show Spots option a discussion of the spot gallery is provided in Section 5 7 below When the data table and spot gallery are both open spots removed from the analysis by clicking on the appropriate check box in the data table are identified by red crosses in the spot gallery Similarly data can be rejected by clicking on a spot in the gallery the data table being updated accordingly Note Please note that for data containing a large number of spots the construction of the data table and spot gallery may take some time A progress bar will show activity in this case 289 Andor Technology Andor iQ USER GUIDE Image Review File Plots Data Data Review File Plots Data View Cell No Frame Area Perimeter Max Chord Shape Int Int Mean Int Loglntint 4 Cel59 15 894 000 134 000 39 000 0 626 66863 000 74 791 11 110 Image CATEMPY Cel60 15 757 000 130 000 42 000 0 563 63747 000 84 210 11 063 Enclosing region i Cell61 15 633 000 118 000 34 000 0 571 36182 000 57 160 10 496 O Cell62 15 1261 000 16
302. fficient i I I 1 I t i I i t j I I i 1 t I t i 1 DO Gamma gt po m 7727 000 Max Invert i g f1600 000 H Min Exclude RE _ ooa Step Gamma Coefficient t 1 j i I I t r I I I i 1 j t I i 1i 1 DO Z Smooth Use Region of Interest Automap Off IV 991 Contrast Map on mouse up 67 Andor Technology Andor iQ USER GUIDE 2 1 1 How the Mapping Table Works 255 Display Intensity output 0 e min Mapping max Image Range Range input To display images of dynamic range greater than the computer monitor we need a method to convert image grey value to display value which is limited to the range 0 255 To do this we use a Mapping Table For display the intensity of each image pixel is converted to a display intensity value based on the transfer function of the mapping table as shown in the figure The Mapping Table form can be manipulated in iQ with the Mapping dialog to achieve an extremely wide range of display effects When combined with pseudo color LUTs look up tables image intensities can be rendered for virtually any purpose Section 4 1 1 2 1 2 The Mapping Dialog Tools In the sequence of figures shown here you can see the uses of the various tools Erm mm 251 000 Mas mm 1067 000 Min available in the Mapping Dialog The figure fio ii Gamma Coefficient TBS VE A S RG roach e FA Poh CAS 1 00 here shows typical histograms from a two im me 7727000
303. fic cellular compartments while photo activation is applied in studies ranging from repair of photo induced DNA lesions to manipulation of light sensitive channels channelrhodopsins http edboyden org 07 03 han html and the study of molecular assembly and disassembly of cellular structures e g actin Phaloblaach and tubulin filaments The techniques can even be Recovery D applied at the cellular level to drug candidates for seen photodynamic therapy arb units C Y An example of a photo bleach recovery sequence intensity profile is shown to the right A represents the pre fluorescence intensity B the bleach depth C the recovery phase where time constant information is revealed and D the recovered level FRAPPA techniques allow measurement of molecular recruitment rates trafficking and turnover in single cells and sub cellular organelles and applications include 135 Andor Technology Andor iQ USER GUIDE membrane and protein binding mitosis and cytoskeleton function In these studies GFP is commonly used in a fusion protein complex to label a protein of interest The bleach or activation phase marks those molecules for observation Quantification of motion or recovery can provide a useful measure of molecular mobility FRAP is used increasingly in analytical devices to determine the identity of unknown substances based on diffusion analysis understanding cellular transduction and identifying li
304. fies the drive where this folder will be located For best performance we recommend a separate fast SATA SCSI or EIDE drive or even a RAID array for this purpose but 429 Andor Technology Andor iQ USER GUIDE only the Administrator can specify the drive However it must be accessible for full rights by ALL users when running Andor iQ otherwise restricted access users will have access only to the Windows virtual memory system the contents of which is lost whenever iQ closes down Image Disk Path The path to the Image Disk folder is configured here when the drive is selected in the drop down box The free disk space of the partition selected is displayed to help select an appropriate place for this folder Note We strongly recommend NOT using the System Partition for ImageDisk as this can cause system operation problems when full A Virtual ImageDisk can be selected which will force Andor iQ to use Windows Virtual Memory System to store images Using the Virtual Memory system will mean that ALL images on the ImageDisk are lost at power down In contrast images stored on a Real Disk Folder will be retained between sessions of iQ unless deleted by the users Private ImageDisk The ImageDisk folder will be created on the specified drive Each Windows Logon User will have a folder for their own images thereby ensuring isolation between different users Shared ImageDisk The ImageDisk folder will be created on the
305. figuration Manager Create a NEW configuration e ea Step 1234 Enter a Name for the Configuration It is recommended that the Name of the Configuration should give some indication of the Devices being used Configuration Name ixon Date amp Time Current Date 2 18 2005 3 09 23 PM Enter a name for the new configuration this should reflect the devices that are to be included in the configuration set up Press Next to continue The second step is to select the camera that is going to be used If you are creating a Fast LZ configuration then you must select the Andor Camera with Piezo Z and Monochromator Monochromator refers to the original Fast L source supported but includes Andor laser combiner and Sutter DG4 And emission filter wheel and multiple shutters can be controlled in FLZ Device Configuration Manager Create a NEW configuration lt lt Back Next gt gt Cancel selecta Camera from the list Select the Dummy Camera Device if you do not have a Camera attached to your system Available Camera Devices Andor Camera vith fast Piezo 4 and Monochromator Andor Camera lt lt lt no Hamamatsu Orca WER 4742 95 Firewire Kinetic Dummy Camera Click on the required camera device and press Next to continue 433 Andor Technology Andor iQ USER GUIDE The third step is to select the auxiliary devices that are to be included in the configuration 43 Device Configuration Ma
306. from the drop down list opens the Software protection Trouble shooter Page 27 Andor Technology Andor iQ USER GUIDE qa Device Configuration Manager File Edit Settings Tools About Exit Software Protection Troubleshooter Read Your Software Protection Key iQ Core iQ Fast LZ iQ MWA iQ MF iQ Analysis iQ Camera Get Donale ID For Kinetic Customers who are Upgrading Existing Software Installing A Second Kinetic Product or g Activating Kinetic Plugins contact Kinetic with this Dongle ID number and you will be supplied with an initailisation code Initialise Dongle Select the Read button to list programs enabled on dongle Select Done when complete 2 3 1 4 Upgrading iQ versions lf you are upgrading from a previous version of the software then your device settings will be retained between versions You should Uninstall the previous version of iQ before installing the new version unless you are installing iQ2 on top of a version of iQ 1 10 3 or earlier If provided please use Andor upgrade tools to ensure backward compatibility of hardware and software When you start to use iQ2 you will notice some differences which we will outline here a iQ 2 will not load an FLZ configuration it will warn you and and close the application or start in Analysis mode only b Whenever you select a Configuration iQ 2 will function with your existing Protocols Channels and Scans from the configuration but you will need
307. from within the series Use image frame number along operating dimension will subtract an actual image as opposed to a single value A value of zero corresponds to the first frame Using another image Use separate image allows a different image from the images being operated on to be used for background subtraction It must have the same dimensionality as the images being averaged When this option is selected the Select button is used to load the image from the ImageList hence it is a good idea to load this image into iQ before running the Episodic Average plugin 342 Andor Technology Andor iQ USER GUIDE PETENTE Switching to the Photo bleach Compensation tab shows the M Along time dimension in each image dialog left Selecting either For all images using the first one as reference checkbox will enable photo bleach compensation in the resulting image Selecting Along time dimension in each image will normalize the intensity of the images through the series along the time dimension When this option is checked the greyed out section below it becomes available allowing the choice of whole image normalization or normalization using the currently selected ROI in the Image Window Selecting For all images using the first one as reference will attempt to normalize all images in all dimensions using the first frame in the series as reference When this option is checked the greyed ou
308. g CTRL or SHIFT while using the left mouse button to select items Once the images are selected press the Go button to create a new image that will appear at the top of the ImageList with the extension ave to show that this was created with the Episodic Averaging plugin Selecting this image and clicking Show will load it into the Image Window Two additional operations can be performed if desired Background Subtraction and Photo bleach Compensation 341 Andor Technology Andor iQ USER GUIDE Episodic Average Switching to the Backg rou nd Subtraction tab shown above there 6 Use contan ae 0 e are four separate methods for C Use mean of area of interest number i correcting for background intensity in some way f Use image frame number along operating dimension O i Use separate image E Subtracting a constant value by selecting Use constant value of and entering a value in the text box to the right this value will be subtracted from each pixel in the resulting image Using a ROI by selecting Use mean of area of interest number drawing a ROI region of interest on the image and entering the number of this ROI in the text box the mean of this region will be subtracted from each pixel in the resulting image When this option is selected an additional option appears below it allowing either the mean of the maximum value in the ROI to be used Using an image
309. g Dell T3500 but has been phased out by some Control Type manufacturers Fortunately you LPT Connections can readily obtain PCI and PCle to Parallel port IEEE 1284 interface cards at a very low cost Examples below have been tested at Andor and showed robust behavior 497 Andor Technology Andor iQ USER GUIDE A StarTech PEX1P PCle Parallel Adapter Card 1 Port SPP EPP ECP B Ultra U12 40736 PCI Parallel Adapter Card 1 Port EPP ECP IEEE 1284 Bi Directional 1 5Mbps 1 If you are using an Andor workstation or other PC with motherboard parallel port then you can simply connect to the port in hardware and software and then start using it See step 6 for more details on use 2 If you use a PCI or PCle card make sure the correct LPT driver is installed and the LPT port shows in the Windows Device Manager without errors In this case you will also need to note the address of the card and set up the Devices ini file in iQ s folder in the Application data path You can check the card status by looking in the Device Manager at the COM and LPT section as shown below This shows the Motherboard device LPT1 and a PCI card m Computer Management d af m File Action View Window Help 8 x es Am Te 2 m zaga m Computer Management Local DGSWDM4 A ih System Tools Computer ia a Event Viewer S Disk drives Shared Folders a Display adapters z Local Users and Groups
310. g Ins Help Set up calibration settings lt lt Back Next gt gt Cancel Stage Calibration o Please selecta slide with a small high contrast feature such as calibrated _ graticule bright field or fluorecsent bead Focus on a feature of interest Move the stage to ensure that the x and y axis are aliqned correctly The feature is defined by clicking ona pixel within it When the feature is moved repeate the procedure to get the new position This will help to define the field of view in stage coordinates Make sure the correct calibration is chosen to match the objective being imaged This allows the system to make the correct sized moves when it is computing the camera angle relative to the stage 128 Andor Technology Andor iQ USER GUIDE RII me icixi As this process proceeds you will be asked File Image List Settings QC Wizards Process Analysis Plug Ins Help to rotate the camera or the stage On an Setup Wizar xY Calibration inverted microscope the latter is not Setup calibration settings possible so the camera may need to be adjusted So be prepared Click on the Next button lt lt Back Next gt gt Cancel Stage Calibration Please selecta dry objective for calibrating the stage The higher the objective the better the precision Calibration 897_20X 08 un iv In this step of the stage alignment iQ will
311. g QICam 4 65 with a circle here the list of cameras Qimaging Micropublisher 3 4 iQ will show this dialog You can enter and then select a camera from the list Camera Type Luca MegaPixel 8 IQ provides We suggest that you keep the alisa short because it is used supported in iQ is automatically populated by iQ along with their pixel sizes 109 Andor Technology Andor iQ USER GUIDE Now you have a list of cameras as shown here and you can complete the Calibration process with one more click on Next reete Prase r Luca Megorval 80O ca Here is the updated calibration list we have created In this case we chose the 887 as our camera in use Ere A 60 0 0 2667 oF ase e fos 42155 ear TOS Min 110 Andor Technology Andor iQ USER GUIDE 3 2 1 Binning Binning is not fixed in the calibration settings The calibration settings are automatically adjusted to reflect the binning chosen during acquisition 111 Andor Technology Andor iQ USER GUIDE 3 3 Differential Spinning Disk DSD Calibration Wizard The Andor Revolution DSD is a spinning disk structured illumination microscopy scanner or SD SIM Because it uses a structured illumination technique it requires computer processing of two image halves to create a single confocal or epi fluorescence result The image processing required is simple enough either weighted addition
312. g further set the DSD processing module of iQ into Debug Mode to get more instrument control Follow these steps 1 Close iQ open the File Manager Go to the folder drive Documents and Settings All Users Application Data Kinetic Imaging Common Drive will normally be C the system drive but may differ in multi boot systems In the Common folder you will find two files as shown below Common File Edit view Favorites Tools Help ae i A i Back gt i Search Wey Folders Address C Documents and Settings All Users 4pplication Data Kinetic Imaginol Common e Go Links x Folders Devices ini El oy My Computer A ouran Settings p s Local Disk Co 0 Andor idi KLINI 9 Config Msi a Configuration Settings E 2 Documents and Settings a 6 KE C3 admin Administrator E CO All Users E Application Data Adobe Andor 4 Andor Bioimaging Apple C Apple Computer 9 FLEXnet Google CO Intelligent Imaging Innovations E Kinetic Imaging O Common Configuration Hie CO ix Microsoft Research In Motion SalesLogix Seagate 559 Andor Technology Andor iQ How To Documents 2 Open Devices ini for editing with Notepad P Devices ini Notepad File Edit Format view Help kinetic Dummy Camera Parameters Camera Type s VOR Syrnc 0 Binning Factor xel Binning Factor Y 1 Black Leyel 128 white Level 128 Brightness 128
313. gand binding sites 3 6 5 FRAPPA Calibration Wizard The following sequence provides step by step instructions on how to calibrate the different FRAPPA devices that may be included in an Andor system Note that it is possible to use more than one device in a system Refer to Section 3 6 6 for multiple FRAPPA device calibration 3 6 5 1 FRAPPA device Before you start running the software make sure the FRAPPA is correctly installed and aligned Here are some tips on the installation procedure but if the unit is already installed then skip to step 3 below 1 Couple the FRAPPA unit to the microscope C mount and if present the CSU Using a spirit level ensure that sure the FRAPPA camera and other components are level and are adjusted to the correct height The system should be mounted on an optical table or breadboard for best results and once the components are aligned vertically make sure to fix adjustable feet etc Note Incorrect vertical alignment will lead to significant errors and may frustrate alignment of the FRAPPA unit 2 Horizontal alignment must now be adjusted This can be done with transmitted light imaging if desired Focus a specimen and adjust for Kohler illumination Sliding the components left to right may be sufficient for alignment and should be done so that no vignetting occlusion of the camera field is observed Once this is achieved then clamp components onto the optical table so they are now locked down Using
314. ge Disk Default Images are NOT retained after shut down Recommended for Komet Users Select a Drive if you want Images to be retained after shut down Recommended for iQ Users An ImageDisk folder will be created on this drive Important An NTFS format hard disk drive which does NOT host the Windows Operating System will improve performance Total Disk Space 9 77 GB Free Disk Space 5 72 GB ONTUNANUOCNCNNMEONNE D C Private ImageDisk Each user only has access to their own Images Shared ImageDisk All users will have access to the same Images Images in RAM Physical Memory usage increases with this value 65536 Some image processing and visualisation operations e g 3D View Clearview amp Orthogonal View require all frames to be loaded simultaneously therefore this setting must exceed the total number of frames being loaded The Default Value is set to 65536 Multi User Access ON the controls will be applied to Users according to the local machine status decided by the Administrator If you have Multi User Access OFF all users have Full access This option is set by clicking on the appropriate checkbox in the Configuration dialog shown over The ImageDisk Configuration is accessed by selecting Global Configuration Settings item from the Settings Menu As previously mentioned all Andor Bio imaging applications use a folder to store acquired image data in a RAW format The Configuration Manager speci
315. ge Positions 4 2 by 2 J t eS Positions 3 Repeat 9 umin 11 planes Se J Move Channel GFP PS ee i ee EA 61 Andor Technology Camera Brightness 128 Camera Contrast 128 Camera Binning CURRENT Camera Binning Y CURRENT Camera Exposure Time 2 0 004 LUT Green Map Min O Map Max 293 Trigger Pre Aa XA SS So Post Analyzer 17 Fluo Filter 7 Fluo Filter 2 Fluo Filter 3 Fluo Filter 4 Fluo Shutter 1 Current Nikon TI Fort 1 TIRF 0 TIRF Angle Trans Filter 1 1 Trans Filter 2 O Trans Shutter 1 Current FRAFFA Curent FRAPPA Owell Time 40 FRAFFA Repeat 1 LYPAT DELTA_ 0 Sutter Shutter Shutter 1 Current xe gt Mi Fe to Andor iQ USER GUIDE Live Move To Adjust Camera Delta Z Go live with that channel all hardware devices will move to the settings specified for that channel Adjust the hardware settings for that channel Adjust a new Z position an offset which allows you to correct for chromatic effects in the optics and acquire the channel at a different Z positions for any reason Acquire Acquire an image with the hardware devices set up as specified by the channel Edit Allows for editing the channel and brings up channel manager Insert Insert a new keyword below this move channel The Tree Insert dialog containing a list of valid keywords for insertion will appear to allow the user to choose Delete Deletes the cha
316. ge file Microsoft Windows AVI media files A multi TIFF time sequence A time series captured using third party software iQ supports a wide range of file types which can be loaded into iQ for analysis Images must be loaded into iQ before Spot Analysis can be performed Further details concerning supported file types can be found in Section 2 4 Images are loaded using the iQ file menu as discussed previously in Section 2 6 1 and Section 2 9 1 Alternatively the Image Window menu can be used Further details regarding these functions can be found in the iQ user guide in Sections 2 6 1 and 2 9 1 respectively A typical open file dialogue is shown below which is applicable to an individual image file or a Open Fil x multi TIFF time Miia pa Bes Look in A Tem sequence ookin C Tere ce Dark OBE tif NEWAN THERSCe LAMBDA tif ES Dark SBiit tif ES NewChrom _B tif ES Aada ES Light 2Biit tif E NewChrom _G tif ES Light 6Bit tif ES NewChrom Merged tif ES Light32Bit tif E NewChrom A ES LightoBittif ES Radars 6 tif File name Files of type Cancel coroa For a time series captured using third party software the Multi Load file type should be selected as shown below 276 Andor Technology Andor iQ USER GUIDE Look in E SBit E c time shutter 5us_Sbit_1280x1024 seq001 time_shutter 5us_Sbit_1280x102 E time_shutter 5us_Sbit_1280 1024 seq002 Fa
317. ge image sequences In Andor iQ the ImageDisk is either shared or held in a separate folder for each Window s User and is located on a Drive specified by the Local or System Administrator during system configuration In this way users data are isolated from each other leading to improved security and performance When you first run iQ a directory called ImageDisk will be created on the root of specified hard drive The ImageDisk is non volatile and so images will be retained between logon sessions In general you will obtain best performance if you use physically separate disks for IlmageDisk and the operating system Windows For optimum overall performance please refer to Section 2 3 6 Data is managed by Andor iQ s Image manager which allows you to treat multi dimensional image data as a single data object similar to a movie clip Thus you can capture review save to disk in TIFF or multi TIF file format and analyze large image sequences with great ease and at high speed On a more technical level ImageDisk provides a virtual memory system which co operates with Windows own virtual memory system Windows system is optimized for general computing so when it comes to images its performance is slow and unpredictable ImageDisk interrogates Windows prior to virtual memory operations disk swaps and ensures that for image operations it maintains free system memory by swapping frames in and out of RAM depending on usage This e
318. ge the dark regions tend to be more of interest so the Dark Features checkbox is selected Lastly Background Subtraction is selected from the dropdown menu and the Preview button pressed to see a preview of the operation in the Rolling Ball Filter window Rolling Ball Filter fe Dark features Background Subtraction v fe Result Radius 130 Background Subtraction v Diso Bright feature Original Image Processed Image Preview completed Here we see that a radius of 30 has been entered and Dark Features selected After pressing Preview the result can be seen in the Processed Image section This is probably an appropriate radius for this image as the salient features have been retained whilst removing the uneven illumination across the field Andor Technology Andor iQ USER GUIDE Rolling Ball Filter Dark features rT En oo Radius 5 Bright feature Background Subtraction FA Original Image Processed Image Preview completed In the example above single molecules can be isolated from the background by using a suitable radius and selecting the Bright Features checkbox After adjusting the Radius and setting it appropriately for the image the Snap or Run buttons can be used to create a single image or operate on all images respectively 362 Andor Technology Andor iQ USER GUIDE 6 7 Threshold Image This tool enables a bina
319. ges from the Image List Disk Info Exit Edit Comments Clicking on this button opens the following dialogue Image Comments C DOCUMENTS AND SETTI BX This is the comments field where you can add any information regarding the images conditions specimen etc This will be kept with the image so long as it remains in the image list or is saved in ABD multi tiff or single tiff formats Conversion to other formats may lose this information Ok Cancel Comments can be typed into the text box as illustrated above and are saved in the image header by clicking on the OK button The comments associated with any image can be viewed by scrolling down the Image Details field in the Image Manager They are stored in an image header field named Comments Text here Comments End For more information on the image header Metadata see Appendix 4 49 Andor Technology Andor iQ USER GUIDE 2 5 3 The Settings Menu To aid in desktop management iQ allows you to select the functionality to be displayed Clicking on the Function option enables selection of the functions that you wish to be displayed Page tabs will be displayed for all selected functions in the iQ Window Function Hot keys Interval Setup Max Auto Time Que Options Functions select the tunctlons required BGB Analysis 4 Calibration w Movie Editor v spot Analysis O Cancel The Max Auto Timeout setting allows you to specify
320. ging These Settings Effect Existing Protocols m Settings COMA control Port CON 7i V Reconnect At Star OK Cancel Pe __ Cancel Status Not Connected Reset 475 Andor Technology Andor iQ USER GUIDE A3 6 2 The Ludl Settings Tab Current device settings can be stored to disk by pressing the File Save button Settings can be imported using the File Load option There are four sections included in the Ludl Settings Tab Position Hardware Camera Ludi Settings WheelA Settings Position Establishing Communication Reset Hardware 7 M Stag Contol Fiter Wheel F 1 217 3 4 M Zmotorcontro Shutter ve 2h of A Position is located at the top of the Settings Tab This will show the current readout of Settings the stage position Control Port COM Status Not Connecte Reset 2s eeenoet ft Chart Feconnect St Stet The Hardware section is under the Hardware Stage Control Filter Wheel fe if 2 sf 4 P zZ motor control Shutter wp 2p a 4 the box for each option being used Up to four different filter wneels and shutters Position part of the Settings Tab Check can be used along with stage control and Z motor control as shown For each one selected a tab is added to the Setup Wizard page Current device settings can be stored to disk by pressing the File Save button Settings can be imported using the File Load option 476 Andor Technology Andor iQ USER GUI
321. gram button and select a parameter to chart as Statistics mee ae Histogram Data eee data ve i dicta Min 504 Save Histogram Data ATA described ie Pr e To am em cose C AA previously sp fe77 an Update Display Sljcene igo CV 64 4 fee Get erate iia A 1 Somber Spots 102 57 SetDefaults Pint graph Pes ae Time resolved data All Data Animate Through Time fe Anitnete Belay UGS z erame j C Statistical plot M tiean Sb Ditties m m C Surface Plot in Number In addition to charting results for the whole data series data from individual frames in a time series can also be visualized using the Animate Through Time option 307 Andor Technology Andor iQ USER GUIDE Variations in spot statistics over time may be visualized with the aid of the Animate check box an appropriate speed for the animation selected using the Delay feature Histogram of Area The animation function can also be fie Data FER Animation accessed from the menu bar at the top sil GEL f e Animate Through Time of the histogram window via the Plot By Statistical Plot Surface Flot option The Animation menu is used to toggle the animation on and off Histogram of Area File Data Plot w On a Be Des Pt CIO Results from any e amet time point of Gs ee es ne es interest can also gf be selected using p the Frame spin BE a bu
322. gy Andor iQ USER GUIDE As with the Rectangular regions ellipsoid regions can be moved resized and deleted with the Pointer or Hand button Right clicking on the image or a selected ROI will also bring up the same menus as discussed in Section 2 10 1 2 10 11 The Polygon Button Olojo S The Polygon button will allow you to define a user defined region on the image for analysis They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and will be drawn in the corresponding color ROPs assigned to a feature are numbered sequentially in the order that they are drawn on the image Click the mouse button at the position of the first vertex of the polygon and then click in turn to define the positions of all subsequent vertices Click the right mouse button to complete the construction of the polygon Polygon regions can be moved and deleted with the Pointer or Hand button but not resized Right clicking on the image or a selected ROI will also bring up the same menus as discussed in Section 2 10 1 2 10 12 The Freehand Polygon Button Hlololss The Freehand Polygon button will allow you to define a user defined region on the image for analysis They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and will be drawn in the corresponding color ROPs assigned to a feature are numbered sequentially in the order that they are drawn on th
323. he 1O CORTO V Mdo Winen o E ETEN AN 41 20 Foncion TADS aioa e eaaa a eaaa aea eaaa iaaa aaaea aa Eaa 56 2T The Image Window ese AA RR O SA A EA AE EEEa 66 2 0 The Image Window Menus oc cocoa seeks cen AU ATU DU AA A TU TA AA AA TUA AA REA E AA AA ERSEL ARa 71 29 The IMACS Nays ator A AA RR AS 86 210 The Mase VOOM AN TARA AA E E E 90 3 CALIBRATION AND ALIGNMENT TOOLS uuuaa sana ana a aa aa aa aa aa RR aa RR RR DURE RRA DURE RR DURA RRA DURA AR Dun ana a Dn DAS 101 3 1 Wizards Help Setting up Your Imaging Systeim ccccccsssssssssscccccsssssscsscccceccssssssccccsssessssssccsssseees 102 3 2 Calibrate Your Syste oseane e E aaeanoa aial aa ea iiai 105 3 3 Differential Spinning Disk DSD Calibration Wizard seecsocssssccecccsssseecoocossssececossssseceeosssssscecossssssee 112 3 4 Dual Camera Image Alignment ecssssssccceccccssscceccossssccceccossscececcssoseececcosssscccecosssscececosssesceeesssssseceesssssssee 119 3 3 SAC ANONIME INE 133o ada AU AT aeaaee TA NU RUTA ES E Ta da AA ESO eae aa aE EU ea an aiaiai 125 3 6 ERAPPA Devices and CaliDrationn sonica oa a a a 133 4 ACQUISITION TOOL S einer eenaveceeacadesavensscueeceussessesecessesvadeeesssvesevedscdescceessenecks 150 2 Andor Technology Andor iQ USER GUIDE 4 COOOMEEUE ALIONS ena cei E A ciate danced cata avec sw easel aa sia sits issuer chase aides SAAD cases DA 151 4 2 Channels and the Channel Wizardsissa a ASU sen caassbanscnestincsasmaacccaassanesnans 1
324. he 3 axis In the 306 Andor Technology Andor iQ USER GUIDE latter case this can be helpful for viewing timed events in a 3D Kymograph but restricted to orthogonal slicing With Z as the third dimension we see an extended focus image of our living or fixed specimen as shown above When you view a volume at large angles you will see gaps between Z sections in the data because we load only the head on view for faster performance If you want good rendering at all angles then use the 4DView from the View menu which is essentially identical except it used XY XZ and Y Z views of the data for better rendering at the cost of speed The dialogs and controls behave in a similar manner 5 11 1 FastMIP and 4D View Options and Animation settings FastMIP and 4DView have options for visualization cropping movie animation creation and image update when XY dimension is changed in the Image Window Zoom is coupled to the mouse wheel while holding down the mouse left button and dragging the cursor left right and up down will change the angle of view on the data set The tools to control FastMIP are explained in a message box when you click on the magnifying glass with query at the top right of the dialog The dialog left shows Rendering Options Include Intensities Between zZz W Activate Grid TY 256 00 g access by click on the 0 00 Resolution Gear button Here YOu Render Method In i 0 urn Maximum Intersity Rendering optio
325. he maximum intensity pixel occurs Same XY position in the dimension and Range selected Range works in the Same manner as described above for Average E g These Encoding tools are useful for creating a Max or Min transmission or emission wavelength map in a spectral image series They can also be used for topographic surface mapping in a Z where the pixels are replaced by the Z coordinate where they appear brightest or dimmest or are in maximal focus In time series analysis feature tracks can be time encoded at each location and the local slope of a track in time will show instantaneous speed Minimum Encoding This is an encoded projection in which each pixel in the Result is replaced by the dimension coordinate at which the minimum intensity pixel occurs same XY position in the dimension and Range selected Range works in the Same manner as described above for Average Delnterlace This process is normally applied to data from RS170 or CCIR analog video images These types of cameras used for many decades in CCTV produce an image by scanning two fields which consisting of the odd and even lines of the image sequentially and then displaying or outputting them for display on an analog video monitor The frame rate of these cameras is 30 fos RS170 US Standard and 25 fos CCIR European Standard but the field rates are 60 and 50 fields per second respectively This means that two fields are acquired per frame and th
326. he rendered image with the Image Window Zoom tool Use this setting for correct Zoom in the AVI output Note This is only displayed in the Saved Movie not while animating in Play List mode Increment This specifies which frames to include in the animation An increment of one will animate through all frames a value of 2 will display every second frame etc Multi channel For multi channel images the resultant AVI movie file is saved as 24 Bit image data If the check box remains unchecked only the first plane in the multi channel data set is saved to disk Use Current Region This option enables only image data lying within a user defined rectangular ROI to be saved as an AVI movie file Save Overlay This option enables Text and Time stamp over lays to be exported with AVI s After specifying the images to include and their parameters Play List mode lets you play the movie using the animation buttons moo Po E Frame The buttons from left to right are Stop stops the animation Jog Back rewinds the animation 1 frame Play animates through the Play List Jog Forward advances to the next frame 271 Andor Technology Andor iQ USER GUIDE Loop animates through the Play List as does the Play button but on reaching the end of the animation it repeats starting from the first frame Bounce continually plays the animation forwards then backwards and so forth You can also animate through the Play List by dragging t
327. he scrollbar The current frame number is displayed in the Frame box The Play List can be saved to disk by pressing the Save List button from where it can later be reloaded by pressing Load List To save the animation as an AVI Movie press Save List as Movie You will be prompted to select a filename and path for the Movie You will also be asked to save the Playback Frame Rate for the movie i e the speed of the movie in Frames per Second Finally you can specify the movie compression AYI File Save a a The saving of AVI Movie files is illustrated Select playback frame rate for the AX file in the series of screenshots shown below 20 Frames sec Cancel Specification of compression decompression algorithm aka codec to Intel MUV codec Microsoft RLE Configure use for movie compression Microsoft H 263 Video Codec About Microsoft H 261 Video Codec Full Frames Uncompressed The choice of compressor is dependent upon which codecs are installed on the system For example on a default Windows 2000 installation the Cinepak Intel 970 Andor Technology Andor iQ USER GUIDE Indeo R3 2 and Microsoft Video 1 codecs are available Alternatively Full Frames can be selected which will not compress the frames Be aware however that files exported in this way could be very large and compression may offer a better size quality payoff for portability The table below summarizes the m
328. how long that a timed iQ dialog will show before it closes and takes default action These dialogs are intended to allow unsupervised actions like shut down in the event of power failure editing settings after a Load Protocol action and so forth 50 Andor Technology Andor iQ USER GUIDE A comprehensive description of iQ functions and page tabs is provided in Section 2 6 Clicking on the Hot Keys menu item enables function keys to be assigned to any channel previously defined using the Channel Manager A maximum of six keys can be assigned during any iQ session Assign Function Keys SAS Pressing a function key will change the current channel to that specified by the hot key If the Force Fe Livevldle z Live when Pressed checkbox is enabled a live Nea i video image will be displayed in the Image Window F4 Run l l using the appropriate image acquisition settings Fh Reserved Fb None HI Pressing the OK button exits the Assign Function F7 None Key dialogue The Reset button returns all the hot Fo None ss keys to the None state no channels are associated FY None ss with any of the function keys F10 Reserved F11 None sts F17 None o o Three of the function keys are reserved and cannot be assigned to a channel The F1 key is used to Force Live when Pressed l access Windows Help information and the F5 key is O Undo used to refresh the iQ Image List 51 Andor Technology Andor iQ USER
329. ialogue is displayed similar to that used during the creation of new device configuration files Section A2 2 The edit function allows modification of the configuration name and active auxiliary and camera devices A2 1 5 Deleting an Existing Configuration t Device Configuration Manager File l Settings Tools About Exit Edit Configuration Delete Configuration New Edit Delete Current Configurations ee Select the appropriate configuration file name from the Current Configurations list displayed in the Configuration Wizard Section A2 2 for further details The chosen configuration can be removed from the Current Configurations list by pressing the Delete button or selecting the Delete Configuration item from the Edit 136 O Andor Technology Andor iQ USER GUIDE menu A Wizard dialogue is displayed similar to that used during the creation of new device configuration files Section A2 2 Before the selected configuration is removed from the Current Configurations list the user will be required to confirm that they want to delete the selected file xi The selected file name to be deleted will be X E E ee S displayed within the lt lt gt gt brackets as shown Are you sure you want to DELETE this configuration in this example lt a Press the Yes button to delete the configuration Clicking on the No button cancels out of the file deletion process A2 1 6 Managing Old Configurati
330. ic Average hich split the image field into two or more regions fara which split the image field into two or more regions sad spit according to wavelength separation of the emission light ace image Image Filter from the microscope The splitting technique allows Rolling Ball Filter Threshold Image simultaneous multi wavelength emission imaging and is Clearview Deconvolution useful for FRET e g CFP YFP or GFP RFP ion imaging Image Math e g Indo 1 co localization and other multi channel Ratio Image o Image Image Math dynamic imaging The technique avoids any delay between Dual Image Disk image channels and is therefore most applicable to live specimens but can be used with fixed samples Optical devices of this kind provide a selection of wavelength by insertion of user defined filters or polarizers One class of devices is designed for single detector use such as the Optical Insights DualView Cairn Research OptoSplit and Hamamatsu Photonics W View Each offers various benefits and options Another class of splitters is designed for use with two detectors such as the Andor DPC Because splitters are purely optical devices they are not under iQ control but they are placed in the optical path between the microscope top or side port and the camera The image split usually occurs in the X axis and consequently the resulting image resolution is half that of the camera resolution in X but full re
331. ile Edit Settings Tools About Exit Kinetic Configuration Wizard New Edit Delete Exit Current User Mark r Current Configurations Test dummy camera Xa Meteor with Kinetic Trigger i Created By Mark Date Created 17 06 2003 12 28 38 o Date Last Modified 17 06 2003 12 28 38 Acquisition Device Standard Video with Meteor Driver File KIDStdGrabWithMeteorll ocx 0 KITriqgerln acx 26 Andor Technology Andor iQ USER GUIDE The available configurations are listed in a Tree View each comprising the following information Created By The name of the Administrator who was logged onto the Local Machine when the Configuration was created Date Created The date the Configuration was created Date Last modified The date the configuration was last modified Acquisition Device The Camera Device used in the configuration The Camera Device Driver OCX used by the Camera Device can be seen by expanding this part of the tree Auxiliary Devices The Auxiliary devices that are contained in the configuration are listed Auxiliary OCX Files The Auxiliary Devices Drivers OCX s used by the Auxiliary Devices can be seen by expanding this part of the tree This is useful for troubleshooting 2 3 1 2 Global Configuration Settings For Details on Global Configuration Settings see Appendix 2 2 3 1 3 Software Protection Key Selecting Software Protection Key
332. ill achieve the same result Show this button displays the image Double mouse click in the list will also show the selected image Rename Image Simply type the new name into the text box and press OK The name of the image will be updated in i the Image List You can copy and paste Enter new name for image to the edit box here Cancel Save Image When a single image is selected this button opens a standard Windows Save File dialogue in which you can save the image to disk When Multiple Images are selected then you are provided with an option to delete an image once it has been successfully saved Browse for Folder Ei ES Select folder for saving images Before the file save operation can Seo HG _bin commence a folder must be selected H Ampdio ef AGM2002 Screen Shots for storage of the multiple image set been Coalescence ae ERER Demolmages After clicking the OK button the save E 1386 MA exa process commences and a message Na Kinetic appears at the end of save operation E KometD ata El Multi Load O NDIMDAta Multiple File Save x AA Ootilas Coot Gequence di PE O All tiles saved successtullv Cancel 48 Andor Technology Andor iQ USER GUIDE Edit Comments File this menu item opens the following dialogue Peon This provides an alternative route for saving image data and Delete Selected Images Delete All Images deleting ima
333. ill allow the orthogonal view to select automatically the nearest Z slice to the focus during the creation of the animation independently for each time point 304 Andor Technology Andor iQ USER GUIDE CT ae eS Clicking on the Rectilinear lv XZ Options Rectinesr _Arimaton show Anim p zy Sre button opens a panel that gives you the possibilty to select a 3D ROI X Y and Z of the rendered image 41 38 45 55 69 89 With an image containing enough dimensions it is possible to create an animation of the orthogonal slices Clicking the Animation button generates a new image containing the orthogonal slices for all time points The animation dimension can be selected in the option panel All currently applied settings will be used to generate the animation rectilinear section display mode intensities inclusion zoom factor and best z slice A window appears during the process to indicate the progress of the creation of the animation Once the animation is finished clicking the Show Anim button will show the newly created image in the Image window The Snap button takes a snap of the currently shown orthogonal slices 300 Andor Technology Andor iQ USER GUIDE 5 10 The Strip View Tool The StripView tool is accessed from the Image Window View menu and is included to provide another way of viewing multi dimensional data In the Strip View you can create a view of a time or z sequence as if it
334. ill be included The Index column is used by the software and does not require any user input There are three possible options for the Background parameter ROI lf a region of interest is defined on the image using one of the region defining tools in the Image Toolbar the average intensity of the region will be taken as the background intensity of the ratioed image Image A dropdown displays all images from the Image List Select an image the background intensity of the merged image will be set to the average intensity of the selected image None The background intensity will be set to zero Each node should be assigned a Colour Response grey for a grey image or a red green or blue component Note Although you can include as many Images as you like in the Merge there are only four colors available to which they can be assigned The minimum and maximum values of the mapping or image contrast should be specified in the Map Min and Max Map This modifies the display of image pixel intensities in the Image Window and is used to improve the contrast of the image or to view specific image properties Only the Image Window display is changed the underlying image pixel intensities remain the same For Multi dimension images each image can be seen and mapped independently There is also a provision for XY jog or shift to allow precise alignment corrections The maximum value can be up to 2 1 for an n bit image e g for an
335. ill open as shown on the right If Display Current Image Slice is selected a live preview will show in the window Press Cancel at any time to exit setup The new image will then be added to the image list Once the processing is complete the new j i r W Display Current Image Slice Destination Image will appear in the ImageList from TTTITTTTTT TT TT Tit where they can be shown and selected for other processing Cancel O Andor Technology Andor iQ USER GUIDE 6 11 Ratio Image The Ratio Image Process enables rapid and precise post processing of single and multi wavelength images from specimens loaded with fluorescence indicators These include ratiometric indicators such as Fura2 for Ca concentration measurements and donor acceptor e g CFY YFP coupling for FRET fluorescence resonance energy transfer imaging as well as normalization techniques for single wavelength intensity modulating indicators lon concentration and FRET interaction measurements can be directly determined from images in a simple yet comprehensive user interface Measurements can be performed on single plane and multidimensional image data The example image below shows a triplet of HEK cells loaded with Fura2 which were stimulated with acetylcholine resulting in temporal fluctuations in calcium ion concentration courtesy Paul Thomas Department of Pharmacology University of Cambridge Ratio measurements can be carried out on images th
336. ill provide Andor camera drivers automatically but third party camera drivers need to be ordered separately and have a separate license iQ will need to reboot after installation to allow Windows to load and initialize the drivers 13 Andor Technology Andor iQ USER GUIDE 1 4 How to Use this Guide We recommend you use this guide as your primary local help The iQ Help menu will launch an Adobe Acrobat view of this document which can be navigated by bookmarks that allow you to drill down into the specific sections you need We have tried to produce a useful text but please feel free to suggest improvements We suggest you read chapter 2 of this guide or there is an alternative iQ Quick Start Guide stored in location C Program Files Andor Bioimaging Help especially aimed at analysis Before you start imaging with iQ please refer to chapters 3 and 4 Finally when you want to Analyze Process or Visualize your data then refer the relevant sections As aides memoires or quick reminders we have provided some How To documents following Appendix 4 These provide very brief step by step guides to achieving certain goal like creating a Channel or a Protocol loading or saving an image in various formats The user guide is arranged as follows Chapter 1 provides useful information on product access overview of installation use of the user guide imagedisk technology and some notes about other related software and
337. important that the LOCAL Administrator runs iQ first and sets up the hardware device settings By default in Win2000 XP all Domain Users are treated as restricted users on the local machine iQ allows Domain Users full access to the Software e g to be able to create edit and delete Protocols and Channels A Note to Administrators To find out more about this go to the Windows 2000 XP Start Menu Settings Control Panel and Select Users and Passwords and use Windows on line help To Summarize the Access Levels provided in Andor iQ depend on Local Status and are Summarized in the following table Access Levels in Andor iQ with MUA ON Log On Status Full Configuration Full Local Admin Y Local User N Y Domain Admin N Y Domain User N Y Domain Local Admin N Y Domain Local User N Y 22 Andor Technology Andor iQ USER GUIDE 2 2 Starting iQ 2 2 1 Andor iQ Logon and Configurations When the user logs onto Windows either on the LOCAL machine or onto a Domain then the access level to iQ features is set When you log onto iQ the program recognizes the current Windows user as is evident in the Hello UserR and Hello Administrator greetings below All Users have access to the configurations list and one is chosen to initialize the current session Registration 3 x Hello Userk you hawe restricted access to the software Flease select the Configuration for this session Hello Administrator
338. in 3 phases and refers to the Master and Slave iQ systems running either on two PCs or two instances of iQ running on a single PC The alignment procedures apply to both scenarios 1 Align mechanically First undertake basic mechanical alignment with a spirit level and make sure the two cameras are in focus 2 Select most frequently used objective Complete steps 3 6 for both cameras this is the fine mechanical alignment phase If using a microscope select the objective you are using most for dual camera Camera Alignment operations This phase will adjust the cameras alignment to within a few pixels Note that due Setup Wizard Camera Alignment Define Feature of Interest to the beam splitter the images from the two Bis Intensity threshold range cameras assuming the same physical i i A lt lt Back Next gt gt Cancel orientation will be flipped about the vertical Ee ze Camera Alignment axis Adjust the camera as required the points will automatically be tracked 3 Select 2 spots for alignment USING aoa scween Points Degrees 15 77558 transmitted or fluorescence light make sure you have a suitable Live Image on each system from the cameras use a specimen with punctate features e g pollen or multi spectral beads The exposure time does not have to be the same but the intensities should 363 0 140 0 188 0 125 0 Point 1 Paint 2 be matched clos
339. in A The user can Load and or Save Settings by BO Threshold A O selecting File on the Menu Bar Select Background B About to view product information 396 Andor Technology Andor iQ USER GUIDE 6 12 1 The Source Tab S OUNCE Background Mask Operation Add Y Diadic Source Operate within N dimensional Image A Runnings UM Select Operator Dimension OF Interest fe Time A 177 B 509 f Operate within N dimensional TIFF f Operate on two images or image seres f Operate on two images or image seres taking average of secondary f Selt Operation taking average of selected range for secondary 6 12 1 1 Operation Operation a Select the Operation from the dropdown list Add This is used to produce a resultant image whose pixel intensity at any coordinate is equal to the pixel intensities of two input images added together at the corresponding coordinates Subtract This is used to produce a resultant image whose pixel intensity at any coordinate is equal to the pixel intensities of one input image subtracted from the pixel intensity of another at the corresponding coordinates 397 Andor Technology Andor iQ USER GUIDE Multiply This button is used to produce a resultant image whose pixel intensity at any coordinate is equal to the pixel intensities of two input images multiplied together at the corresponding coordinates The result will be a float
340. individual pixel intensities to ionic concentrations The progress dialog shown here is displayed as the processing may take a little time On completion of the calculation the resultant ratiometric image is displayed in the Image Window W Display Current Image Slice PTT TTT Cancel 6 11 7 Analysis of Temporal Data The ratiometric time series can be studied in the Analysis Window and statistics calculated for individual time frames or within user defined regions of interest ROIs The use of the analysis functions available within Andor iQ has been described previously in Section 4 6 Results from the example image series are presented below Three images are displayed together with statistical plots showing the mean brightness computed from corresponding user defined ROls positioned at three different locations on the ratiometric image Examination of the statistical plots reveals a significant difference in the dynamics of the Ca distribution Andor Technology Andor iQ USER GUIDE Statistical Plots O O ta OO taf F gay te tel fap et ab ee leh CADA fel O O O E TOMO Nr ODORrFROM TON e O O TO A OW TF ON See Lith lyst te ptf CS eS 40 60 50 100 120 140 160 180 200 20 Statistical Plots Ooooo ogoooo ooqaoococaoaoaoooeom8ewmUmCcUw8lUcO UO NETO O O ON Hw TONer O O OLA O ALE ON NULA S o oe o o ape e e e o 40 60 so 100 120 140 160 180 20 Statistical Plots 260 240 220 200 180 160
341. indow during OpenGL operation and or pollution of the animation with overlapping dialogs remaining in the rendered result Because of these possibilities we recommend you visit your graphics card website and download the latest drivers wherever possible More recent systems supplied and tested at Andor do not exhibit these problems 308 Andor Technology Andor iQ USER GUIDE 5 12 Montage View The Montage tool is companion to Montage scanning explained in some detail in Chapter 4 The Montage tool is designed to stitch together a series of frames from overlapping fields reconstructing them into a larger field of view image In order for Montage to complete this task iQ stores in the image meta data see Appendix the position coordinates of each field of the Montage Montage uses this information to adjust the overlap between frames and then merge them into a result See examples below The image above shows a Montage of 48 8x6 fields 20 X magnification from a Molecular Probes sllde of a mouse gut section Tiling artifacts are visible 309 Andor Technology Andor iQ USER GUIDE This image shows a Montage of 24 6x4 fields 60X oil 1 4 NA objective using the Revolution XD laser confocal system The field images comprise 30 Z planes and 2 channels each and were stitched with Montage and a maximum intensity projection MIP produced to show this result 5 12 1 Montage View and Dimensions The Montage View tool
342. ined Statistics are calculated for the entire image The calculated statistics are displayed in the chart The Settings button opens a dialogue in which you can specify what region of color space to consider This is described in Section 5 3 1 Note In order to access the Settings button the Threshold option must be ticked in the Options tab Pressing Analyse will perform the analysis on all regions defined on the image using the ROI tools if no regions have been defined the analysis will be performed on the entire image The results calculated depend upon the statistics selected on the Options page Section 5 3 4 The data calculated from the analysis is displayed in the table Each row contains the statistical data for one user defined region of interest 259 Andor Technology Andor iQ USER GUIDE The following statistics are calculated Area the number of pixels within the region within the threshold limits specified in the Settings dialogue Area the percentage of the area within the threshold limits specified in the Settings dialogue Integrated Intensity the integrated intensity within the threshold limits specified in the Settings dialogue Integrated Ratio the ratio of the intensity of one color component compared to the intensity of another component e g the RedGreen Integrated Ratio XI Red I Green Minimum Threshold the lower pixel intensity threshold limit specified in the
343. ing point image where pixel values are not scaled but represent the real product Divide This button is used to produce a resultant image whose pixel intensity at any coordinate is equal to the pixel intensities of one input image divided by the pixel intensities of a second input image at the corresponding coordinates The result will be a floating point image where pixel values are not scaled but represent the real quotient of the two Source images 6 12 1 2 Diadic Source Operate with an N dimensional Image The values under the Source tab will change depending on which radial button is selected e Operate within N dimensional TIFF f Operate on twa images or image seres f Operate on two images or image senes taking average of secondary f SelFOperation taking average of selected range for secondary If Operate within N dimensional TIFF is selected Select an image to use Diadic Source Operate within dimensional Image a A This will default to the himngsUMT S Select image currently in the Image Window Press the Select button to choose a different image This will open the Image Selection Window Double click an image from the image list to select 398 Andor Technology Andor iQ USER GUIDE Operator Dimension OF Interest lf Time is selected select a value from ie f Time each dropdown list 5 If Operate on two images or image series is selected Select which Operation to u
344. inning Use Current A Wait Allocation On Ex DA Protocol loaded the Device Setup tab shows a new tab Camera Isl Camera Selection Use Current Channel Pause Click OK to start protocol Selection in which the loaded O Interval 5 5 ms fastest Scan a5 Repeat 36000 times cameras are listed Using the Channel Chi Device Setup l n Snap controls on this The user can D End l Loop Protocol l V Display image Edit the Alias user defined name and the colour of the Save As Image Window background as a Options visual cue to remind you which Ui camera is loaded The Device setup Camera tab is shown over the page with the Camera Selection tab 4 7 1 2 Load Protocol and Camera Selection To switch between cameras under Protocol control you can set Protocol1 to use Camera and set Protocol2 to use Camera2 And when you want to run the two sequentially add the Load Protocol action at the end of Protocol 1 and you can apply the same approach to use multiple protocols as well each loading the next In this way you can switch operations between different imaging modes with different cameras on different microscope ports a powerful mode of operation 220 Andor Technology Andor iQ USER GUIDE Acquisition Auxiliary Devices aaa a 221 Andor Technology This screen shot shows the Camera Selection tab in the Device Setup Dialog
345. inside the DSD so there will only be a need to switch this control if a different set of fluorophores is to be used Acquisition Auxiliary Devices ES co TE EEA Andor AMH Andor DSD Prior Lumen Andar AFZ Control Settings DSD Filter wheel GFP nm Contocality signal f High Signal f High Sectioning sectioning 492 Andor uses light sources see next section with excitation filter wheels faster switching than the DSD unit for rapid multi wavelength imaging In that case then the multi band DSD filters will be held in place while the excitation wavelength is switched for faster operation The second control on the DSD interface allows you to choose between High signal and High Sectioning The spinning disk used in the DSD utilizes a structured illumination pattern SIP combined with Andor Technology Andor iQ USER GUIDE image processing to achieve optical sectioning This contrast of Signal to Sectioning defines which pattern will be used during in imaging The lower frequency provides more signal since it has a broader axial Z response while the higher frequency SIP provides stronger sectioning or narrower axial response So selecting the relevant control on the GUI moves the disk to ensure the correct SIP is used to modulate the specimen illumination 493 Andor Technology Andor iQ USER GUIDE A3 11 Metal Halide Source Prior LumenPro Andor iQ controls a range of light sou
346. interactively using the Image Importer Wizard 542 Andor Technology Andor iQ How To Documents A5 10 How to Import Multi dimensional Images Part 2 1 Select Image Importer from the File menu 2 Using the J button browse to the drive and Select Common Name directory of the files to be imported Image Importer Wizard Image Import Options Next gt gt Cancel 3 E nte ra name th at iS common to al th e fi les to Firstly you should select a directory that has all the image files you want be imported Click Next Image Importer Wizard Image Import Options Set Import Dimensions 4 Now the to load in then type in a common name that is contained by the files You can also specify a name for the destination image Finally click the Next button to go to the next page Common File Name Destination Image Name SIMULATED Importedimage Directory F Multiple Images EI Files dimensions of SIMULATED PSF D004 t lt lt Back Next gt gt Cancel SIMULATED_PSF_z0005 tif v the imported tee eens to check on those dimensions that the resultant image must be specified In this case the image If you have selected Multi Channels you also need to choose the number of channels for the image Import Dimensions M Maulti Channels SIMII ATFD PSF Minne tif lt 5 To complete the process enter the unique dimension identifier for each dimension In this case z will s
347. into other Windows applications such as Word Paint etc 2 8 2 2 Edit Event Markers The Edit Event Markers item enables the modification of image event markers The edit dialogue displays a text box containing the event marker name together with information identifying the dimensional location and associated frame number 72 Andor Technology Andor iQ USER GUIDE Be Al Event marker information is store Event Event Mame Event Information subsequently stored with the Iw interrupt 1l Frame 33 15454 ms image by enabling the 2 F 50 19692 f ea appropriate Store Event Moa Frame 77 26492 ms check box and exiting the OK button Iw 5 Frame 110 34702 ms WE Frame 130 46372 ms Mo Frame 170 56492 ms Enter names forthe events OK On exiting the dialogue a message box is displayed prompting the user to confirm that events with no name are to be deleted from the list Edit Event Markers i x as P There are events For which no name has been entered Do you wish to delete these events Cancel Store Event Ewent Name Event Information ra Interrupt Frame 33 15454 ms Enter names forthe events OK 73 Andor Technology Andor iQ USER GUIDE 2 8 2 3 Add Event Marker The Add Event Marker item allows additional image events to be added to the event list SES Please selecta name forthe New Event Marker linterru pt 2 Frame 142 49412 ms C
348. ion is requested demanded 7 Setting the FRAPPA focus control Now that the FRAPPA system is calibrated in X and Y you can use it in FRAPPA on Demand mode This means you can go live and use a FRAPPA region type with a FRAPPA on Mouse Up action Place a FRAPPA region on the focused specimen image and right mouse on the region Select the FRAPPA on Mouse Up option when selected it will be checked Assuming the calibration above is accurate when you mouse up after moving the region the area immediately below the region in the specimen will be bleached It is possible that the spot size will be larger than desired this depends on the exact optical configuration but in general it should be possible to bleach a spot of lt 1 0 micron If the spot size is much greater than 1 micron or appears to be in another optical plane Z position then you can carefully adjust the fiber input focus position To do this remove the laser safety shroud see above and you will find a thumb screw which when rotated moves the fiber position in and out Adjust the position in small increments while using the Mouse Up action to bleach Once the spot size has been reduced to 1 um or so then you should consider this a good setting If the beam cannot be reduce to about 1um then you may need to reduce laser power or bleach time 140 Andor Technology Andor iQ USER GUIDE Note Make sure to replace the laser safety shroud if you
349. ions A comprehensive description of the Plug In functions currently available may be found in Section 5 2 54 Andor Technology Andor iQ USER GUIDE 2 5 8 The Help Menu Help selecting Help from the file menu will give you three options ner First selecting Help from the menu opens the iQ Online Help Configuration Product Activation file About 1 iQ Status Selecting Product Activation from the stss u F Help Menu opens the Status window z Posti coe O la lf product does not have atime imit From here you can see the available S fitos AE GU ay Camera This product does not have a time limit Andor iQ modules A tick mark ine Si nds AE This product does not have atime limit indicates activated products The right P Jeteduct ia analysis This product does not have a time limit side of the dialogue shows which A ae IS PIOOUCK OOS MOC Aaxve Ume nmk modules are activated and the existing Product i_CleatView This product does not have a tine limit Product iQ Tracking Refresh eer This product does not have a time limit time limit if any select the Refresh button for changes to show Please contact Andor Bio imaging division for further details on activating iQ Modules Andor Bioimaging formerly Kinetic Imaging Product i Pressing the About button Version 6 03 29 displ inf b h d BuildTime 16 59 35 Feb 17 2005 ISplays in ormation about th
350. ions give the resultant image C Set Background to 0 into the text box default is gnare pixels belor background Image ame Create The resultant image will be displayed in the Image Window and added to the Image List lf Use current region for normalization is selected you should define a region about an unchanging area on the image using the ROI tools in the Image Toolbar The mean pixel intensity of the ROI is calculated for each frame of the image and the intensity of each pixel in that frame in the image divided by that value Depending on the sample and whether the fluctuations are localized it may be possible to use the entire image intensity as the baseline with which to normalize In this case ensure that Use current region for normalization is deselected The mean pixel intensity of the entire image plane is calculated and the intensity of each pixel in that frame divided by that value lf the data needs to be background corrected a Background ROI s should be defined using the ROI tools and the check box enabled for the Background Correction will use Background regions option A background intensity corresponding to the mean pixel brightness of the ROI is calculated for each frame of the image and pixels with intensities less than or equal to the background intensity can be set to zero or ignored during the subsequent calculation 414 Andor Technology Andor iQ USER GUIDE The choice of the pa
351. iption box set the NA a 1X 1 Define the objective parameters 7 P oe Lens RI defaults to glass 1 515 and the 1 5 1 5 o 3 immersion medium air water or oil and 2 5 2 5 4K 4 Select the objective magnification from the Next gt gt ca S85 10x 10 list that iQ pop up when you click on the Objective Definition 16 16 SS button to the right hand edge of the Lens SS Magnification box which is highlighted T with an elipse in this figure Description 60 60 ricer mmesin Bins 100 100 Objective Lens 150 150 Lens Magnification x 250 250 60x 60 Avs Numerical Aperture Immersion medium 1 2 Water ET Lens Refractive Index 1 515 E Next gt gt Objective Lis Mes Ns 10 0 0 3 11 515 None 0 3 17 515 None 1 515 Oil 1 515 1 515 1 515 1 515 None Oil Oil Add Edit Remove Lens RI lmm Type Description peal a SE Water Cancel 1 0 1 0 1 515 1 0 1 33 1 515 1 515 108 When you click Next you return to the Edit Objective List dialog and the new objective is added to the list Repeat this sequence until you have completed your list Once the list is complete click on the Next Button and you will then been shown the Camera List box Andor Technology Andor iQ USER GUIDE ccBack Netos FEA The Camera dialog shows the currently selected camera in this case an Andor Came
352. is continually evolving please contact Andor Technology or your local agent to confirm support of new devices We are currently developing the Andor iQ 2 stream of releases With iQ2 Fast LZ has been removed This enables iQ2 to provide a single unified user interface with one set of acquisition Protocol rules Protocols flexibility and performance in iQ2 has been greatly extended so that many more sophisticated acquisition procedures can be performed For more information see Chapter 4 The acquisition engine for iQ2 is being incrementally multi threaded and its performance will soon match that of FLZ For existing users who require FLZ performance we have maintained backward compatibility with iQ 1 10 IQ2 also offers the Python interface The Python IDE is an open source module available free of charge from Andor Technology and it provides a user programming environment which enables extension of iQ and an enormous resource repository of open source libraries See www python org for more information We are delighted that you have decided to use Andor iQ and hope it will prove both 8 Andor Technology Andor iQ USER GUIDE powerful and easy to use Python IDE Integrated Python Image blurring and haze Development Environment reduction Embedded batch queve for Deconvolution of 3D 4D and 5D script execution data Support for data exchange with Imaris and ImageDisk Push and pull data from i to ImageJ Ha
353. is is the section at the bottom of the Window If the values do not reflect those entered type the values again A3 6 4 The Z Tab Enter the Device Resolution x Stage Z Settings microns step Z The Default value is shown in the picture below The Pee odie on default for open loop Z motor 7 0000 operation on the Ludl is 100 nm per step or 0 1 micron step Operating closed loop the motors are commonly set to 50 nm per step or 0 05 micron per step Closed loop operation uses with a linear encoder gauge to monitor relative position of the stage and objective The linear encoder is attached to the microscope objective turret with a special bracket and the encoder reference plate is attached to a fixed plane on the instrument Note The user must press Enter after changing the Z value Verify that the Value entered was set by checking the Establishing Communication Section at the 478 Andor Technology Andor iQ USER GUIDE bottom of the Window If the value does not reflect that entered type the value again You either hit the Enter key on your keyboard or click on the Accept button to ensure this value is set A3 6 5 The Ludl Filter Wheel Tabs You can choose up to four different Hardware i Stage Control Fiterwheel fe IT 27 oF 4 Filter Wheels from the Settings Tab PE Shute Aea Camera Ludi asi Wheel A Settings A common configuration is to use one wheel for a Excitation and another for Emission
354. is time to start imaging Andor Technology Andor iQ USER GUIDE 6 4 Flip Rotate The Flip Rotate Tool is accessed from the Process menu Episodic Average Clicking on the selected item displays the dialogue shown Mask Wizard Field Split below Flin Rotate Image Image Filter Rolling Ball Filter Threshold Image Clearview Deconvolution Best Z Image Math Ratio Image Image Image Math Dual Image Disk Flip vertical Flip Horizontal Rotate 90 Rotate 180 Rotate 270 The Flip Rotate function enables any image displayed in the Image Window to be reoriented to one of five different geometries by clicking on the appropriate button Images can be flipped about the vertical or horizontal axes or rotated through 90 180 or 270 in the clockwise direction This tool is useful for correcting feature geometries prior to analysis or reorienting images for publication 355 Andor Technology Andor iQ USER GUIDE 6 5 Image Filter The Image Filter plugin can be found in the Process menu in iQ s Main Window and provides some common image filters such as averaging and edge detection When opened the Image Filter window will appear with a preview of the currently displayed image as shown below Filtering iQ Image Filter Average v Repeat 1 Original Image Select the desired filter from the Filter dropdown menu Filters available are Average Lowpass Highpass X Highpass Y 2D Lapla
355. isk controllers 33 Floppy disk drives ES IDE ATAJATAPI controllers a9 IEEE 1394 Bus host controllers or Fae ee oe The above picture shows the appearance of Boards in the Device Manager after installation of board and drivers from iQ installation procedure After restart select My Computer on your desktop right click mouse to select 460 Andor Technology Andor iQ USER GUIDE properties Then select Hardware Device Manager You should be able to see the card listed there as shown 3 Acquire Card Number e After PC restart run InstalCal from Start gt All Programs gt Measurement Computing e InstaCal will automatically detect the card then a board number will be given as shown below lt gt 154 Bus Hae Bd O DEMO BOARD HES Bd 0 DEMO BOARD lt a gt PCI Expansion bus 2 dido PCI Expansion bus 4 Bd 1 PCI DIO24 dev 2 Bd 1 PCIM DDA06 16 dev O The above picture shows the card installed as Bd 1 in the Instacal initialisation program Exit InstaCal 461 Andor Technology Andor iQ USER GUIDE Acquisition Auxiliary Devices TE Camera Trigger Input Output Set Up 4 In Andor iQ the Local Machine Administrator Uses Device Setup Controll Board Bdtt 1 PCI DI024 Acquisition Auxiliary Devices Pa m Snap Snap i Record E Configuration with the Manager tamea Till Monochromator After you have built a to use PCU or Fa
356. ki Ng on the Histogram of Area rl File Data Plot Options Statistical Plot Mean Maximum button provides an Moum alternative means of data presentation Mean Min Max and SD vales SE can be plotted as a SS pa a function of Mm E ys scsi dees e 155 E frame number 5 a Vedere Dip adda Stilt Charting the Se fe ay Pigon A Vn wee number of spots versus frame number is also possible by enabling the Number check box The Statistical Plot option can also be selected from the Plot menu and the parameters to be plotted selected from the Options menu Histogram of Area Histogram of Area File Data Plot Animation File Data Plat Options All data v Mean e Animate Through Time van 8 Statistical Plot m aE Surface Plat 3 400 ie Min os Number 3 200 309 Andor Technology Andor iQ USER GUIDE When the Save Histogram Data button is pressed the selected statistical parameters e g Mean SD Max Min and Number are written to a data file together with a summary of the histogram statistics The Print Graph button enables the displayed statistical plot to be printed for further inspection and comparison An example Print Histogram window is illustrated below save and Print functionality is also available from the menu bar as described previously Print Histogram Iof x The example Printer WEB SERVER Brother HL
357. l number of your Camera Selection Camera Piz PIXY controller listed Control Settings Other options are NONE not connected or Demo to enable simulated controls without Status Not Connected having a PI controller device connected Device Select NONE PI C 86 Piezomotor Controller SM 0111155825 MICRON F anns Notes on the usage of the PI XY Z devices e Supported models of Physik Instrumente XY stages require stage registration every time iQ is started up Please refer to Section 3 2 of this document for details on the stage registration process e Section 4 3 of this document describes how to create and edit XY scans and Z stacks to use the XY axes in a protocol See also appendices A5 2 and A5 3 for How To guides to creating a Multi Field scan and a Z stack 502 Andor Technology Andor iQ USER GUIDE e ANDOR jQ Appendix 4 Image Metadata 503 Andor Technology Andor iQ USER GUIDE A4 1 iQ 1 X Image Headers and Metadata A4 1 1 Introduction This document presents the structure of iQ 1 10 image headers section by section Examples will be provided when necessary The header is constructed like a Windows ini file which is managed via section headings and endings denoted within square brackets e g section section1 end Within these sections we provide relevant information about the image size dimensionality position data and acquisition condition
358. lable until this procedure has completed Click Cancel to exit without registering the stage Or Complete the following sequence 1 Click Start to move to the Home position 2 The stage will then return to the original position Please ensure that there is no chance of damaging optical components during SSE SLI at 4 Please ensure that there is no chance of damaging optical components during movement Ok to continue Cancel to stop Cancel Cancel NOTE If your system has a motorized stage then you will see the dialog above when the system starts up if the stage has been moved or powered off between sessions Make sure your lower the objective lens to avoid damage and allow iQ to calibrate your stage This will ensure your scan patterns are repeatable Andor iQ has a Calibration Manager which provides two modes to calibrate camera pixel and system optics to specimen dimensions for quantitative analysis 105 Andor Technology Andor iQ USER GUIDE The first is Automatic and relies on knowledge of the optical magnifications in use and the pixel size of the camera chip We will briefly describe this mode here The second is Interactive and requires use a calibration device such as a graticule or haemocytometer with the selected objective ox File Image List Settings QC Wizards Process Analysis Plug Ins Help The Calibration Manager guides you Setup Wizard through creating calibrations It
359. lation IF vou wank to review or change any of your installation settings click Back Click Cancel to exit the wizard IMPORTANT Please remove all USB SuperPro keys before continuing Installshield Cancel The Sentinel Driver installation will complete Windows NT users may have to re boot their PC after driver installation 420 Andor Technology Andor iQ USER GUIDE A1 2 Troubleshooting Dongle Issues lf the Andor Software runs in Demo mode even after plugging the dongle onto the parallel or USB port and installing the Sentinel Drivers described above there are a number of things to check The drivers are installed correctly Start gt Settings gt Control Panel gt Add Remove Programs F Add or Remove Programs Of Currently installed programs sort by Name ad Change or bana ee Microsoft MET Framework 1 1 Size 3 07MB 3 E Microsoft Firewall Client Size 6 19MB 5 i Microsoft Office 2000 5 1 Professional Size 157 00ME Add New 5 NISTime Programs ES PowerDVD Size 1 73MB EN Primess Size 299MB eens Ce QuickTime Size 2r 2MB Se ES RadLinker Size 1 62MB Components Sentinal SEE E 26 d rarely Set Program SS To change this program or remove it from your computer click Change or Remove iE Shockwave dA SmartFTP Size 3 61MB p Symantec Antivirus Client Size 14 1 9MB A The Proxomitron Ver Maoko 4 4 Size 1 98MB El Tweak UI a Winamp remove only i Windows
360. layed in the Image Window having pixel intensities equal to the pixel intensity of the first image plus the pixel intensity of each subsequent image at the corresponding coordinates Merge O k Resultant Image Name Mergelmage Image Wavelength nm sel ci Mergel U None Menges U None Green 0 255 Menges U None Blue 295 Meret 10 ree ines Gres 0 Create Preview i d Delete Load Save Press OK to close the Merge dialogue The current settings will be remembered the next time that RGB Merge is selected from the Process Menu On completion of the Merge the resultant image is added to the Image Manager It is assigned the name typed into the Resultant Image Name text box The Image column is used to select the images to include in the merge To select an image click on a cell in the column A dropdown list will display all the images currently in the Image List from which you should select the required image Note All images must be the same size along each dimension The merged image will be displayed in the Image Window having pixel intensities equal to the pixel intensity of the first image plus the pixel intensity of each subsequent image at the corresponding coordinates The second column in the table is used to specify which frames to include in the merge In this example only the frames that have been acquired at wavelength zero A10 Andor Technology Andor iQ USER GUIDE w
361. lculate stage X and Y resolution as a sanity check for the stage calibration This may take some time e g 10 seconds The cursor may flash during this process This is normal behavior This last panel contains a summary of your new settings left It provides a good sanity check regarding your motorized stage calibration You can indeed compare the result calibration with the resolution set in the Device Setup dialog in the stage panel and update the latter but please make sure that you check with the stage user guide or our support team to confirm the correct setting Note that an error in the Calibration Wizard section 3 2 such Andor iQ 1 10 0 Andor Camera With Fast z and Wavelen i iojlxj o File Image List Settings QC wizards Process Analysis Plug Ins Help as INCO rrect specificatio n of th e C mou nt Setup Wizar Av Calibration magnification can give rise to scanning setup calibration settings errors when pixel to stage movement is incorrect lt lt Back Hest gt gt Cancel staqe Calibration Summa s Resolution Pixels Actual Distance Calibrations E 06 483 ileal clea 0 08 Y Resolutio Pixels Actual Distance Calibration f 05 066 f 260 9999 132 Andor Technology Andor iQ USER GUIDE 3 6 FRAPPA Devices and Calibration FRAPPA was the name we gave to our first innovative photo stimulation product which is described in more detail below FRAPPA is an acronym for
362. lds 32768 9165 000000 15017 000000 124 830000 Fields 32768 8691 000000 15724 000000 124 830000 Fields 32768 9165 000000 15724 000000 124 830000 11252 8966091472 17453 5315027801 133 17 11252 8966091472 18160 5315027801 133 17 12200 8966091472 18160 5315027801 133 17 19224 5387770383 17265 4838432294 120 67 19224 5387770383 17972 4838432294 120 67 21249 7528330019 17202 8012900459 120 67 21249 7528330019 17909 8012900459 116 5 22197 7528330019 17909 8012900459 116 5 9290 36510636712 20015 4281119273 137 33 9290 36510636712 20722 4281119273 137 33 11315 5791623308 19952 7455587438 137 33 11315 5791623308 20659 7455587438 137 33 12263 5791623308 20659 7455587438 137 33 19287 2213302218 19764 6978991931 129 19287 2213302218 20471 6978991931 129 21312 4353861855 19702 0153460095 124 83 21312 4353861855 20409 0153460095 124 83 22260 4353861855 20409 0153460095 124 83 9353 04765955069 22514 642167891 124 83 9353 04765955069 23221 642167891 129 11378 2617155144 22451 9596147075 120 67 11378 2617155144 23158 9596147075 145 67 12326 2617155144 23158 9596147075 120 67 14351 475771478 22389 2770615239 112 34 14351 475771478 23096 2770615239 112 34 16376 689827441 7 22326 5945083403 108 17 16376 689827441 7 23033 5945083403 108 17 17324 689827441 7 23033 5945083403 108 17 19349 9038834054 22263 9119551568 104 19349 9038834054 22970 9119551568 104 21375 107939369 22201 2294019732 137 33 21375 107939369 22908 2294019732 137 33 22323 11793
363. lds are listed under the XY Positions keyword and can be Edited in the Tree by driving the stage with joystick or GUI if one exists to a desired location and then right mouse click and select Record Current If you select Move To from the right mouse Edit menu the stage will go listed coordinates immediately Return to Table Repeat Z lterate through a series of Z planes to create a focus series Specify number of planes n range of motion m um step size Az m n 1 Z Scans are computed relative to the current position Ze as center This makes the maximum and minimum z values Zmax Ze M 2 and Zmin Ze M 2 If you have more than one Z control e g microscope and Piezo stage you can choose which one to use for the scan execution In the XY Scan Wizard you access the Select XYZ dialog and choose the device Return to Table 199 Andor Technology Andor iQ USER GUIDE Snap Frame Acquire a single frame from the camera with the following sequence Open the shutter s with Open Close status preframe action trigger the camera wait for the exposure time Close the shutters with Open Close status postframe action and transfer the data to the ImageDisk This action will communicate a message the Image View to display the frame if the Display Image check box is selected on the Acquisition tab at the bottom of Protocol related buttons Note The timestamps for snapped frames are added as the frames are acquired Due t
364. le File Save window will Multiple File Save be displayed asking if you wish for J Do vou want to delete the images From the image list after successfully saying files to be deleted from the ImageDisk Yes Cancel once they have been saved Clicking esl No to this dialogue would mean they remain in the ImageDisk AND are saved out to another directory Clicking Yes would be a good way of ensuring the ImageDisk does not become too full over time 545 Andor Technology Andor iQ How To Documents 5 Clicking Yes or No will produce a See File Browser window in which the select ramen rar a destination drive and folder is selected Once the destination has been chosen E 8 My Documents My Computer clicking OK will complete the saving of Gi 344 Floppy As See Local Disk Co the files from the ImageDisk The files ge ImageDisk Di See Current Image Disk E will be saved in TIFF format Hs Datav7GB Fi H Removable Disk G thes Images CD Hs Shared Documents 4 Test s Documents Me Mehanrk Blaree 546 Andor Technology Cancel Andor iQ How To Documents A5 13 How to Save JPEG or BMP Images 1 To save the currently loaded image Hamamatsu Orca I ER 4742 95 Firewire AET Edit Image List Region View select Save Display JPEG or BMP omme AXI mfo ele T OLE F01 Ml s ve s Regions Save Display JPEG gt Highlight from the File menu in the Image Window 2 There are fou
365. le task at lower cost and with less sample material Spot has however proven to be of broader application and can be applied to the quantification of the morphology and density or intensity of any population of discrete objects The image analysis allows intensity discrimination thresholding object measurement and filtering inclusion or rejection and statistical analysis and presentation of the population data The results can finally be exported to a spreadsheet for further analysis An image based object review interface shows all selected objects and allows the user to rapidly identify and reject artifacts In statistical plots histograms and scatter plots clusters can be selected or rejected from the analysis Spot functions include Analysis protocol file management Interactive threshold selection Interactive object parameter filtering based on Area integrated intensity and shape factor Batch processing of file lists using user selected protocols Analysis of time series images for studying spot dynamics Numeric and image based object review tools Histogram and scatter plot presentations with parameter range selection rejection Export of selected data for further analysis in spreadsheets 274 Andor Technology Andor iQ USER GUIDE Spot provides a straightforward user interface with powerful interactive configuration and automatic processing of image sets The overall goal being to provide a flexible yet rapid and
366. lected as this will automatically provide a list of positions points which will deliver the best calibration for the device range 4 The Calibration panel allows you to calibrate how the device range translates to camera pixels This ensures that interactive FRAPPA operations can be executed at 144 Andor Technology Andor iQ USER GUIDE the correct position Below the combo box of pre defined device positions if Auto Increment is checked the device will automatically move to the next position when the Add Calibration Point button is clicked for the current position If Bleach On Selection is also checked the new position will be bleached allowing the calibration to flow more easily Now with the specimen in focus select a suitable Frap channel and click the Bleach Sample button If the system has been pre aligned you should see in the image a region of the specimen which has been bleached A bleached region will appear darker than the rest of the specimen and should be localized Note if the bleach FRAPPA Calibration Help Setup Wizard FRAPPA Calibration setup FRAPPA alignment and calibration settings Alignment Summaries Current Frappe Alignment Full auto Mosaic calibration Exit New Edit Delete T Select Current Device w Mosaic FRAPPA Full auto Mosaic calibration cannot be seen you may need to scroll the XY stage a little or increase the FRAPPA time
367. light source Andor iQ ensures that the sample is exposed for a minimum time period by ensuring the shutter opens and closes as closely as possible with the camera exposure commands However there may still be significant fading that must be addressed This is necessary for visualization where fading is a nuisance and also in quantification where fading represents a decreasing baseline upon which fluctuations must be observed Photo bleaching compensation can be achieved using two different methods 1 Normalization baseline adjustment normalize the image sequence by ratio with a Known unchanging area 2 Depending on the sample and whether the fluctuations are localized it may be possible to use the entire image intensity as the baseline with which to normalize A background correction can also be applied to the data Following the calculation of a mean intensity pixels with intensities less than or equal to the background level can be ignored or set to zero during the photo bleaching compensation calculation Problems may arise when the objects in the sample are moving and the ROI is not a valid fading reference 113 O Andor Technology Andor iQ USER GUIDE select Photo bleaching Compensation from the Process Menu Processing will be performed on the current image Resultant Image Name lImageName M Use current region for normalisation Type the name you wish to l Background correction will use Background reg
368. ll be the same length as the ok Cancel Help Original nDim The second method takes a ratio between two image series such as one might load from a third party capture software using the multi load facility in AQM If the image series are not of the same length the resultant image series will be The length of the smaller of the two sequences and the additional frames in the longer sequence will be ignored except where one of the images is a single frame and then the result will be the length of the longer sequence 390 Andor Technology Andor iQ USER GUIDE If one of the Images is a single frame the single frame will be used as the numerator or denominator in all ratio operations loxi The third method illustrated in this File About SS dialog behaves in a similar way to Source Background Mask Calibration Ratio Source Ratio two images or image series method 2 except that the user MNumerato WNETSERVERADEMOIMAGESACA RATIONFURA TIF Select defined frame range in the Denominata WINETSERVERSDEMOIMAGES CA RATIOSFURA TIFE Select Denominator Region OF Interes denominator image will be averaged The average is used as_ the Wavelength l l Q ie denominator for all ratio operations Event f For denominator average images The Dimension selection for Start 2025 End 6504 denominator range will almost always Ratio within N dimensional TIFF be
369. llocation Of Camera Selection Use Current tig Everts dP Pause Click OF to start protocol Se Repeat T 100 times 10 sec Move Channel Ch al Snap Ao Move Channel Che al Snap i End TO 7 Protocol Mult phase MEZT m Image Multi phase MEZT a Protocol Type Time Series ah Camera Binning Use Current Wait Allocation Off Camera Selection Use Current g Events dP Pause Click OK to start protocol gi Repeat T 100 times 10 sec S Repeat Channel Ch1 Ch2 H Channels Description al Snap End Channel i OnLineChart i End TO Py Protocol Mult phase MET p Image Multi phase MEZT a Protocol Type Time Series ah Camera Binning Use Current T Walt Allocation Of Camera Selection Use Current Events dP Pause Click OK to start protocol Se PAepeat T 100 times 10 sec Repeat Channel Ch1 Che H Channels Description al Snap di End Channel i End TO The Online Chart is used to monitor channel intensity in regions of interest during Protocol execution Online Chart can be inserted into the Protocol Tree after the End Channel action as shown below The on line Ratio processing keyword may also be inserted into the protocol but instead of charting ROI intensities it will compute a pixel by pixel ratio between the two channel images To configure the details of processing you can right mouse on the Ratio keyword and
370. llows you to define a user defined line on the image for analysis They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and will be drawn in the corresponding color ROPs assigned to a feature are numbered sequentially in the order that they are drawn on the image Click the mouse button at the either end of the line and then click in turn to define the positions of all subsequent vertices Click the right mouse button to complete the construction of the polyline Right clicking on the image or a selected ROI will also bring up the same menus as discussed in Section 2 10 1 2 10 8 The Define Freehand PolyLine Button The Freehand Polyline button will allow you to define a user mime defined region on the image for analysis They will be assigned to the feature currently selected using the Current ROI button Section 2 10 17 and will be drawn in the corresponding color ROPs assigned to a feature are numbered sequentially in the order that they are drawn on the image Click the mouse button at the position of the first vertex of the line and then drag the 96 Andor Technology Andor iQ USER GUIDE mouse on the image to define the rest of the line structure Right click to end the construction of the polyline Freehand Polyline regions can be moved and deleted with the Hand or Pointer button but cannot be resized Right clicking on the image or a selected ROI will also
371. logy Andor iQ USER GUIDE l5 xi When you have finished creating or File Image List Settings OC Wizards Process Analysis Plug Ins Help Setup Wizard editing your scans they will appear in Scans the Scan Manager list of scans Scan Manager clearly identified as Montage scans as shown here New Edit Delete Scan Summaries Current Scan Large Areas Sm ocan Large Areas FFA Montage Acquisition Region ARR Current Fields 1 og Calibration Scan Linear Montage FAA Montage Acquisition Linear ARR Current Fields 1 o Calibration Select Z Device 4 3 5 3 Montage Scan Review and Visualization When iQ executes a Protocol with a Montage scan within it it will acquire the data and organize into a logical n dimensional structure which can be navigated in the Image Window The Montage dimension specifies the individual fields within the montage An example is shown in the first image on the next page If the Montage is associated with a multi field scan then there will be a Field dimension and a Montage dimension If the Montage is associated with a multi well multi field scan then there will be Well Field and Montage dimensions Montage scanned images have a Montage dimension in the Navigator as shown above which allows exploration of the individual tiles or fields To create a stitched or tiled view of the fields you can use iQ s Montage Plugin
372. m run the program update exe and follow the on screen instructions 425 Andor Technology Andor iQ USER GUIDE A1 5 Installing the Windows Scripting Host If you are running under Windows NT and are using the Andor Image Disk you will need to install the Windows Scripting Host to use the Image Disk Configurator 1 If the installation program is not already running insert your Andor Installation CD Rom into your CD Rom drive and run the program Start exe which is in the root directory of the CD This will start the installation program which runs side by side with an installation Online Help Guide 2 Select the Help and Utilities page and click on the text Windows Scripting Host 3 Follow the on screen prompts accepting the default values The Windows scripting Host will be installed on your system after which you will need to reboot your computer Note You can also install the Windows Scripting Host from the CD Rom without starting the installation program Run the program ste51en exe from the Wsh folder on the Andor Installation CD Rom 426 Andor Technology Andor iQ USER GUIDE A1 6 Installing your Andor Software 1 If the installation program is not already running insert your Andor Installation CD Rom into your CD Rom drive and run the program Start exe which is in the root directory of the CD This will start the installation program which runs side by side with an installation Online Help Guid
373. mage frame number along operating dimension Use separate image Denominator Backgraun C Use constant value of f Use maximum of area of interest number Use image frame number along operating dimension Use separate image Denomata Select Source Background Calibration Numerator Background Region Max 1 Numerator Threshold 0 Denominator Threshold O Denominator Background Region Max 1 Four methods of background correction are available for the numerator and denominator Any method may be selected by clicking on the appropriate radio button Use constant value of performs a background correction by subtracting a fixed value from each pixel a value for the constant is typed into the text box This is normally computed from average background intensity Use maximum of area of interest number subtracts the maximum pixel value in a user defined region of interest ROI from each pixel The ROI used for background correction is specified by typing the required ROI number into the text box 385 Andor Technology Andor iQ USER GUIDE Use mean of area of interest number subtracts the mean pixel value in a user defined region of interest ROI from each pixel The ROI used for background correction is specified by typing the required ROI number into the text box Note The ROI must be placed on the image before opening the Ratio tool or by closing it and then drawing a
374. maging sessions Users will need to manage the ImageDisk to release space when it starts to get full or to Save important data to standard image file formats like multi TIFF Note that Imaris 7 1 now reads the ImageDisk directly and greatly reduce data exchange times between the two ImageDisk performance will suffer if a drive becomes too full 80 or more or if NTFS file format is NOT used In order to check double click My Computer and Right click each Hard Drive selecting Properties from the pop up menu This will show the current file system and free space The ImageDisk in Andor iQ now warns the user when the disk is becoming full and provides a warning at each 5 increase above 75 At 90 iQ will demand the 36 Andor Technology Andor iQ USER GUIDE disk be cleaned up and or backed up In Multi Level Access different Windows users will be provided with a separate ImageDisk folder to ensure that they do not delete or corrupt another user s image data All images are acquired to the ImageDisk and the memory management ensures that when system memory RAM is limited rapid disk transfers are used to buffer images not in use In this way the ImageDisk provides a substantial enhancement to memory management and performance with the large data sets commonly acquired processed or analyzed in Andor iQ ImageDisk access and control functions are controlled from the ImageList menu item 2 3 n Dimensional Images nDims
375. me frames with two corresponding wavelengths of 340 and 380nm respectively The first frame at 340nm is shown above and was acquired using iQ with a Monochromator as the excitation source and a Hamamatsu 4880 80 camera 380 Andor Technology Andor iQ USER GUIDE 6 11 2 Source 5 x File About Ea Images to be ratioed are selected SOUICE Background Mask Calibration within the Source dialogue Ratio Source Ratio two images or image series Mumerato JE BEASTAIMAGES KROMASCAN IMAGESSCHAOM _F TIF Celect Denominato WPURPLE BEASTAMAGESKROMASCAN IMAGES CHAO Select Clicking on the Source tab reveals the panel displayed below Ratio within N dimensional TIFF Ratio two images or image series taking average of denominator C Selt Ratio taking average of selected range for denominator Background Mask Calibration Numerator Background 320 Numerator Hone Threshold 0 Denominator Threshold 0 Denominator Background 325 Ok Cancel Help Four ratio methods are accommodated by the ratio tool and are selected using the appropriate radio button e Ratio within N dimensional TIFF e Ratio two images or image series e Ratio two images or series taking the average of the denominator e Self Ratio taking the average of the denominator 381 Andor Technology Andor iQ USER GUIDE The method selected determines how the image processi
376. me fs 0 04 Pe LUT Grey wizard will take you step by step through the creation of the channel displaying information to help the user at each step The first dialogue in the Wizard will ask you to enter a name for the channel Subsequent dialogues in the wizard will depend upon the hardware devices included with your system Detailed tutorials for setting up channels for different types of Experiment are included in Chapter 4 in the User Guide 31 Andor Technology Andor iQ USER GUIDE To edit a channel select it in the list and press Edit or use Device Setup You will then step through the wizard as with setting up a new channel except you will not be asked to name the channel setting up your hardware devices NOTE Because the channel will be available to all users for a specific hardware configuration it is important that you have a clear naming convention For example we recommend the following Prefix Channel names with a lower case letter for the mode and use upper case for the fluorophore E g vVGFP means visual GFP for switching to the eyepieces for user viewing of data while cGFP means confocal GFP and tGFP means TIRF GFP settings In this way you can agree and maintain clean and robust system setups Copy allows you to create a new channel having the same setup as an existing channel Select from the list the channel you wish to copy then press the Copy button A dialogue will pro
377. mension by 1 step 367 Andor Technology Andor iQ USER GUIDE Select the best focused slice for time 1 and press the Select Z button Continue to select the Best Z for each time point in the time dimension Once all Best Z selections are set Press the Create button and this will create a new image on the image disk which has only one Z point for each time point as shown in the figure below Best Z psf_dtimes display the new image in viewer Selected 2 21 Selected 22 Time 2 Selected 2 21 Time 3 Selected 2 22 Time 2 3 Time 4 Selected Z 22 S 56 f Time Time 5 Selected 23 Time 6 Selected 7 22 Time Selected 2 21 All points done Saaz so Undo Create Clear All Close Note You can use the Clear All button at any time to start over completely or use the Undo button to erase the previous step 368 Andor Technology Andor iQ USER GUIDE 6 9 2 Multi Channel Image When using multi channel image with a time series follow these steps select a multi channel image from the image list Select Best Z from the Edit Menu on the Image Window this will start the Best Z with its Channel Offsets Dialog which is different from the single channel cases Use the Channel Selection button to select only the first channel to be displayed in the view Use the Image Navigator at the bottom of the image window to set the Time Tab dimension to 0 Select the Z Tab
378. mpt you to enter a name for the new channel Type the name into the text box and press OK ch2 The new channel will be added to the list where aK Cancel it can be Edited as required Name To delete a channel select it in the list and press Delete Press Close to exit the Channel Manager returning to the Acquisition dialogue 32 Andor Technology Andor iQ USER GUIDE 2 3 3 Scans Scans are created edited and managed in the Scan Manager Scans specify the co ordinate sets for image sequence capture when more than one field of view and or more than one focal plane in the sample is to be observed This requires motorized XY and or Z control Scans are configured through the Scan Wizard and are inserted and deleted to and from Protocols to specify acquisition sequences scan types include multi well multi field and montage types iQ 2 allows insertion of multiple Z scans but only one XY scan Scans specify settings for motorized XY stages microscopes and piezo focus mechanisms They can be re used and edited directly in the Protocol Tree Section 4 3 More options are revealed by using a right mouse click scans are set up using the Scan Manager which is opened by pressing the Scan button on the Acquisition dialogue 33 Andor Technology Andor iQ USER GUIDE File Image List Settings QC Wizards Plug Ins Help Setup Wizard Scan Manager Scans SS Koo e New Edit Delete
379. n Andor s Differential Spinning Disk DSD is a spinning disk structured illumination system with optical sectioning capability In other words it is a confocal scanner which uses computer image processing to achieve optical sectioning The calibration procedure for this device ensures precise alignment of two images of the specimen and should be executed daily or whenever a filter turret is changed See section e Select XYZ Device in some configurations multiple XY or Z devices might be present e g there may be a piezo Z as well as a microscope 102 Andor Technology Andor iQ USER GUIDE 103 motorized focus This wizard allows you to specify which device iQ should control for Z scanning It is important for performance and range reasons a piezo will provide fast control over small movements 100 s microns while a motorized microscope focus will provide relatively slow control over a much wider range millimeters The same comparison is true for other axes piezo XY and stepper motor XY stages can co exist In order to manage multiple z devices iQ 2 1 introduced what we refer to as Reference Z and Scan Z Reference Z represents the slower more course Z device such as a microscope Z motor It is used to associate a Z position with XY movements Scan Z represents the faster fine changing Z device It is used for scanning through a series of z positions This wizard allows you to select the
380. n Appendix 3 Acquisition Auxiliary Devices x P oj Current Live Snap Record Camere Selection Camera Prior DTF Olympus FRAPPA Piezo i Staion Top DU8S7_BV 4708 Settings Exposure General Device Info Error Log lt lt Window Size 512 512 Gai Full Chip Window Binnin M x V E x1 Shutte Open Close Auto Frame Averaging OF M Use Pre Amp Gain EMGainEnabled 2 Exposure Tim Frame Transfer Live fao ms v Display Contro Fast Refresh meee Display Every Frame C Display 5 Frames s Readout ms 30 357 Actual Frame Rate 0 00 Trigger Contro Free Run Ext Trigger Avail Memory MB 2047 Camera Statu Camera is working Tips ye Temperature 70 Do a Timeout 1000 mes 38 Andor Technology Andor iQ USER GUIDE 2 3 9 ROI s and Classification Regions of interest ROIs can be readily defined and subsequently analyzed using iQ s analysis tools e Paula drosophila PHC Verticalklip Rotate1 80 16 Bit 642 0 107 x 10270 0 107 4D Fle Edt ImageLlist Regon View Fast MIP yw ajajaj NN 212 DOlO S T i me ro E Analysis parameters such aS mean pixel intensity peak intensity etc are readily computed for a complete data set or from user defined regions on the image Copy ed ROIs may be constructed Delete 1 1 1 Delete A using any combination of Classify
381. n Auxiliary Devices ES Once the system has been 7 ai Current Record Nett calibrated it is very easy to use Camera Andor AMH Andor DSD Andor APZ The Camera dialog houses a new DR328 1158 Settings DSD Processing tab called DSD Processing as W Enabled Calibrate Alignment i shown here This should be imaging Mode f Confocal C Confocal amp Widetield enabled and then the user can f Wideteld ve M nioc 7 fe RAW select the Imaging Mode as Confocal Wide field or both and this will determine which images iQ v Background Correction Confocal Black Level a captures during a Protocol The Wide field mode can be very helpful in focusing the specimen in the first instance A slider is provided to set the Confocal Black level We recommend that the black level is set by imaging a region of the specimen where there is little no real signal or where the specimen is out of focus and ensure that the resulting confocal grey levels are in the range of a few tens 117 Andor Technology Andor iQ USER GUIDE The following figures show wide field and confocal images of the same field in a three color tissue preparation The high levels of detail lack of flare and dark background is apparent in the confocal image 118 Andor Technology Andor iQ USER GUIDE 3 4 Dual Camera Image Alignment Modern imaging systems can be readily configured for captur
382. n Intensity the Mean intensity value of the selected region and time frame Peak Intensity the Peak intensity value of the selected region and time frame SD the Standard Deviation of the selected region and time frame 262 Andor Technology Andor iQ USER GUIDE Area the number of pixels within the region within the threshold limits specified in the Settings dialogue Section 5 4 1 Area the percentage of the area within the threshold limits specified in the Settings dialogue Section 5 4 1 Integrated Intensity the integrated intensity within the threshold limits Specified in the Settings dialogue Section 5 4 1 olntegrated Intensity the percentage of the integration within the threshold limits specified in the Settings dialogue Section 5 4 1 Integrated Intensity Per Unit Area the integrated intensity divided by the number of pixels i e the area within the region At the bottom of the Options Tab under Channel Selection Channel Selection are check boxes to LI Cho 400mm Solid x select which channels will be graphed IV Chi 385 nm Dash Use the drop down lists to specify the appearance of each channel on the Dash Dot Dot Graph Small Dot 263 Andor Technology Andor iQ USER GUIDE 5 5 2 The Options Page RGB Analysis Tab Acquisition Analsis BGE Analysis Calibration Movie Editar Spot Analysis Analyse Analyse Settings Grey Level Statistics Channel Statistics Coinci
383. n animates forwards through the nDim to the final frame 1 1 When the Normal Mode button is selected on animating the nDim with the Play button forwards or backwards it will animate through the frames of the image once and then the animation will end EF The Loop button animates the nDim as does the Play button but on reaching the end of the animation it repeats it starting from the first time frame AH The Bounce button continually plays the animation forwards then backwards and so forth The scrollbar above the animation buttons can be used to lic scrollbar with the mouse or click on the arrow buttons The stamp value of the scroll through the frames of an nDim Simply drag the current frame is displayed above the scrollbar When animating through an nDim the number of the current frame is displayed in the Frame Number text box This text box displays information only values cannot be input 87 Andor Technology Andor iQ USER GUIDE When animating through an nDim you may not wish to view every time frame in the image It is possible to specify the frames for viewing the remaining frames are ignored To select a sequence of frames the number of the first frame in the sequence is selected in the Min range text box Animate to the first frame in the sequence and press the Set Min button to accept the value The number of the final frame in the sequence is selected and displayed in the Max ra
384. n file are displayed in a Tree View structure in the right hand panel Files are selected by clicking on the appropriate file name using the left mouse button The Tree View is expanded by clicking on the icon The Make a Backup Configuration File each time the Configuration Manager is Closed checkbox shown at the top of the dialogue remains active by default This ensures that back up copies of configuration files are created each time the Configuration Manager is closed The archive facility may be deactivated by disabling the checkbox Unwanted configuration files can be removed from the Old Configurations listing by 438 Andor Technology Andor iQ USER GUIDE selecting a file and then pressing the Delete bution Selecting the appropriate file and clicking the Rewind button activates a previously archived device configuration file Clicking on the Done button exits the Manage Configuration Archive dialogue A2 1 7 Settings Device Configuration Manager File Edit Metsu Tools About Exit Global Configuration Settings Software Protection Key Enable Demo Version New Eat Delete Current Configurations Confic The Settings drop down list is used to edit Global Configuration settings view Software Protection Key Settings and to Enable a Demo Version on the software A2 1 7 1 Global Configuration Settings For Details on Global Configuration Settings see Section A2 1 439 Andor Technology
385. n is sawed will be available to all users so it is therefore important to have a clear naming convention that describes the Muwe T scan setup In this example we have named the scan pattern Costar ssf 3x2 35 39 Model of the micro plate Type of scan systematically aa sampled fields Number of columns and rows 3x2 of wells Well diameter in mm Well separation In mm Press Next to continue 169 Andor Technology Andor iQ USER GUIDE oe Se Select whether you are using a elup Wizard Multi Well Petr circular or rectangular well lf Set up Well settings Circular enter the well diameter in mm into the text box If Rectangular enter the well height 44 Back Next gt gt Cancel and width Vell dimensions Press Next to continue select the type of well and enter the well dimensions f Circular diameter 35 Rectangular i File Image List Settings QC Wizards Plug Ins Help Enter into the text boxes the number of eee ramen eee are et ee columns and rows of wells setup well settings For the Well Separation enter the distance in mm from the start of one row to the start of the next and from the start Back Newt gt gt of one column to the next Multiwell plates Number of wells Press Next to continue Number of columns 3 aa ie Gaal Sumber of rows Well separation Between rows Between columns 170 Andor Technology Andor i
386. n the following format X Y Z Region Info Fields Field This section contains the width and height of a field in um Region Info Fields This section contains the position of all fields including montage sections for 505 Andor Technology Andor iQ USER GUIDE montage scans All values are separated by a tabulation The first line contains the number of fields the number of columns of the following table the version number of the data set and the date and time of when the file was written The second line contains the titles of each column of the table The following lines describe the fields of the acquisition Environment This section contains the user name and the iQ configuration name Protocol Description This section contains an exact representation of the protocol tree Grab Parameters This section contains some camera information Camera camera type Andor for Andor cameras Window Left left of the area of the camera chip containing the result image Window Top top of the area of the camera chip containing the result image Window Right right of the area of the camera chip containing the result image Window Bottom bottom of the area of the camera chip containing the result image Window Width width of the area of the camera chip in pixel used for the result image Window Height height of the area of the camera chip in pixel used for the result image BinningX binning of the X
387. n using Wizard gt Stage Calibration The Stage Alignment Wizard is accessed via the Wizards menu In the first dialog select the Multi well option and follow instructions Select a channel for the camera settings if you have one existing Place a micro plate onto the stage move the first well into position above the objective lens and focus onto the bottom of the well You can use a low power objective e g 10X to make this process as simple as possible Make sure there is some feature to focus upon even a scratch or debris in the micro plate base will do Move the stage in the X direction and make sure that the feature moves parallel to the horizontal axis of the Image Window Andor iQ 1 9 1 Andor Camera File Image List Settings QC Wizards Process Analysis Setup Wiad eS Camera Orientation Multi Well Plate and Petri Dish lt lt Back Next gt gt Pele Plug Ins Help Cancel prea Orientation Steer to the centre ofthe first well 126 F Press the Next button and then find the approximate center of the first well as shown Andor Technology Andor iQ USER GUIDE Andor iQ 1 9 1 Andor Camera xI Now move the left hand edge of the first File Image List Settings QC Wizards Process Analysis Plug Ins Help Setup Wizard well into the field of view of the objective Camera Orientation Multi Yell Plate and Petri Dish Th
388. nager Create a NEW configuration lt Back Next gt gt pa Step 1 2 3 4 Select the devices from the list that you wish to use in this configuration Available Auxiliary Devices CASI Filter Wheel Device _ Atto KIAtto AIC Device Cairn Filter Wheel Device _ Cairn Monochromator Device C Conix ZMotor Device _ CRI MicroColor Device Leica DMR Device Nikon E1000 Device C Olympus X81 BX61 Device CRI LCTF Device Ludi Stage Device Bi Lud Filter Wheel Device Piezo Device _ Marzhauser Stage Device Kinetic Monokromator Device Prior Filter Wheel Device _ Prior Stage Device _ Sutter Instruments Filter Device Till Monochromator Device Kinetic Trigger Device The devices are selected by checking the appropriate check boxes as illustrated in the example above Press Next to continue A summary of the selected camera and auxiliary devices is displayed At this point the user can edit the device selection if necessary returning to the previous pages in the set up wizard by pressing the Back button i Device Configuration Manager Create a NEW configuration lt t Back Finish gt panel Step 1 2 3 4 Configuration Summary ames on CameraDevice fAndorCamera SOS S OOOOO Auxiliary Devices Troubleshooting The Camera and Auxiliary Devices listed above are Registered when finishing the Configuration Wizard To check for succ
389. nalysis In this way regions within regions can be used to created holes and regions within holes can be used to create islands and so forth Figure where all regions have been classified as blue and therefore the enclosures create holes and islands This provides a powerful tool for analysis of many irregular naturally occurring features and structures Region blue Aa Holes blue Islands blue Figure ROIs and Group Analysis 266 Andor Technology Andor iQ USER GUIDE 5 5 4 Background Correction When analyzing fluorescent images the image can be considered composed of bright objects on a dark background In most circumstances the background value can be considered as the black level or an image offset To obtain precise intensity data from the bright objects of interest it is common to correct for this background value by subtracting it from each pixel during analysis The background value is computed from a region within the image and the mean value from the region is used In iQ the background value is computed from the Group of Red regions in the image If you select the Background Correction option then the Red region mean value will be subtracted from each pixel at analysis time Grey At increasing exposure times the background increases slightly while the peak intensity increases more dramatically At longer exposure times saturation occurs and the profile is flattened as displayed above re
390. nce gt gt 488 and 561 two checks clicked and the Burst and Bright Field Control Count 50 Int s 0 000 Z O aan number above is set to the sum of No Bright Field Edit L Control wavelengths 1049 as a further V Enabled Move 1st Number of Lambdas 1 EnabledL M a indication This example is shown Current L G E C 104900 S6 because dual acquisition may require Excitation viv I7 REA 12 Z Emission 500 000 507 000 5200005 simultaneous excitation of two MS EZ is fluorophores for emission splitting Z Control lv Enabled Start 35 8 Current Z ES d evi ces n Move 1st 35 8 Ste A SE setae Note that a Z scan has been specified Computed Param Planes Start Planes Step end FD gt here and the Burst and Brightfield Nyquist Formula for Step Gol step through planes 0 GoCente Control settings at the top of the dialog M Use Nyquist Step 35 80 ed bed ole mi po Time Burst 0 728 s Save to file Load from fie now have different values from the previous Sequence tab illustration above We will deal with this point later when we discuss matching Master and Slave frame count during acquisition Now you should be able to go Live and obtain an image from a test specimen if the system is setup correctly 4 7 2 4 Setting Up the FLZ Protocol First create a Protocol in the Protocol Manager by clicking on New and choose the Fast option as shown since you want to
391. ncident Statistics page Chromaticity color fraction analysis r R R G B i e the red chromaticity value equals the number of red pixels divided by the total number of all color pixels red green blue g G R G B b B R G B 5 5 3 Group Analysis The analysis of regions of interest is a common action in image analysis In iQ regions can be defined in any combination of rectangle ellipse polygon or freehand polygon These types are selected from the Image Toolbar as described in Section 2 11 As regions are drawn they will be given colors according to the specified class of region Section 2 3 9 When analysis is performed regions of the same color will be placed together in the data table but each region will have its own separate statistics in the table However when you have several regions of the same color representing discrete parts of a sample feature or phase you may wish to view the analysis results from the regions as cumulative statistics or as a Group To achieve this you select the Group Analysis option check box on the Options dialogue and then all regions of the same color will be treated as one combined region The data analyzed for all these regions will be grouped and reported as one data row 265 Andor Technology Andor iQ USER GUIDE One further important consequence of the Group option when selected is that regions of the same feature color that overlap will exclude the overlapping area from a
392. nd ROIS 3 6 8 Multiple FRAPPA Devices Andor iQ2 software allows control of more than one FRAPPA device The following sections describe how to setup and select calibrations for multiple FRAPPA devices how to create a multiple device protocol and how to select a required device when performing manual FRAPPAs 3 6 8 1 Multiple Device Calibration The following descriptions assume familiarity with section 3 6 5 FRAPPA Calibration Wizard Open the Calibration Wizard and select the device from the radio list as shown in the diagram below Now run through the Wizard steps as described for the particular device in section 3 6 5 Repeat this process if required ensuring that you have a calibration for each of the devices you need control of 146 Andor Technology Andor iQ USER GUIDE Alignment summaries Current Frappe Alignment Mone Mew Select Exit Select Current Device CO MicroaPoint FRAPPA Mosaic FRAPPA On creation of the calibration the Current Frappa Alignment will be automatically assigned to the new calibration Alignment Summaries Current Frappa Alignment hosaic_260 710 ey Egit Delete Select Exit Select Current Device blicroaPoint FRAPPA w Mosaic FRAPPA mehdasaic 200710 In order to control multiple FRAPPA devices iQ needs to know which calibration to use with each of the devices If you have multiple calibration files for a device you can instruct i
393. ndle huge data sets Andor IQ Core Extensive hardware control filter wheels motorized microscopes confocal spinning disk Optimized for EMCCD technology Extensive and flexible mult dimensional acquisition analysis and visualization Accessible wizards for protocol setup Applications include Time lapse fluorescence microscopy FRAP FLIP lon signalling single molecule microscopy GFP Gene expression Luminescence Immunofluorescence microscopy Tracker Multi well Fully automatic and interactive Capture and analyze data from motion analysis wells of microtitre plates Multi channel image management Control of motorized stages Comprehensive data analysis High content screening options for display of Multi field Drug dose response testing displacement speed and direction F ie Ene Overlay track presentation Capture and stitch multiple fields Applications include all motility Control of motorized stages and single molecule tracking Autofocus to enhance performance over long term studies Andor Technology Andor iQ USER GUIDE 1 2 System Requirements Andor iQ requires the following minimum computer configuration e Minimum Pentium 4 2 8 GHz running Microsoft Windows XP Professional We recommend the fastest possible PC with multi core processor high performance RAM and graphics adapter e Minimum RAM 512 MB 2 4 GB recommended e Fast hard drive for the operating
394. nels as all of this power will be concentrated into a single laser spot during FRAPPA actions 4 Run the FRAPPA Calibration Wizard from the Wizards menu If there are 137 Andor Technology Andor iQ USER GUIDE multiple FRAPPA devices in the configuration select the FRAPPA FRAPPA radio button to calibrate the FRAPPA device Choose to create a New FRAPPA calibration or choose the FRAPPA calibration you want to Edit 5 Next you must centre one of the FRAPPA galvos for imaging as shown in the figure below Select the CSU imaging channel and make sure that you can see a clean confocal image Note this step is not required for Mosaic and MicroPoint FRAPPA Calibration View Setup Wizard FRAPPA Calibration alala akie mlelsis r o X ro I Set up FRAPPA alignment and calibration settings FRAPPA Alignment Adjust the light path using the control below Please indicate direction of image motion XorY Once the image is centered click Next Light Path 6 15 Galvo Pos j Observed Image Motion N x s gt lmaging Channel GFP Widefield o1 4 e zy I Automap 1000 Using the single axis control as shown above set the Imaging Angle to the value shown on the Calibration label this label should be attached to the unit Note that if you are Editing a FRAPPA calibration then the Wizard should reset this value automatically Then change the angle in steps of 0 1
395. new camera if it has different size pixels or if you add an objective to the system 1515 1 0 Default setting This panel here lists the objectives in use E505 1 0 1 515 1 0 1515 Oi 1 515 but you should edit the list to reflect the ESE 1 0 1 515 1 33 1 515 Oil 1 515 Objective List 1 515 1 515 in your system iQ populates a default list objectives in your system by selecting Edit Note that the colors used with each objective ease identification and reflect the standard colors used by all manufacturers on objective bodies Next gt gt cancel In the Edit Objective List dialog you can Objective List i Edit the objective settings with the Edit button a new dialog allows you to enter description NA immersion medium and refective index properties ne EA ii Extend the list by Add a new 1 0 1 0 dialog will provide a list of objectives 1 515 1 0 1 33 choose and then edit for the speific magnifications from which you can 1 515 1515 properties of your objective lil Make sure you Remove objectives that are not installed on your system to avoid confusion for users 107 Andor Technology Andor iQ USER GUIDE File ImageList Settings QC Wizards Process Analysis Plug Ins Help File Ed 5 5 N a Ea IS A OS Here you see the Add Objective dialog Defne Objave o 88605 Complete the descr
396. ng proceeds and the different experimental situations where each method would be appropriate are discussed here When we mention image series bear in mind that this is a times series but can also refer to single frame images ioj x Fie About Method one illustrated in this SOLUCE Background Mask Calibration dialog uses an nDimensional Ratio Source Ratio within N dimenszional Image Mumerato WNETSERVERADEMOIMAGESACA RATIOXFURA TIF Celect Ratio Dimension OF Interes f Wavelength f Time tf Event Numerator 340 Denominator 380 image nDim as its source as produced by AQM in which two channels or wavelengths series are recorded in the same nDim The user specifies which channel is used for numerator and G Balo mihin N dimensional TIE denominator In this case the ratio C Ratio two images or image series C Ratio two images or image series taking average of denominator of 340 to 380 nm IS to be i Self Ratio taking average of selected range for denominator computed In this case note that hlask EAr only one source image is Source Background Numerator Background Region Mean 1 displayed in the configuration graphic This is the classical dual Numerator Hone Threshold O Denominator Threshold 0 wavelength ratio approach Denominator Background Region Mean 1 Because of the capture method in AQM the resultant image series wi
397. nge text box Animate to the final frame in the sequence and press the Set Max button to accept the value The Increment value is used to specify which frames to include in the animation An increment value of one will animate through all frames a value of 2 will display every second frame etc This value can be set either by pressing the arrow buttons or by typing a value directly into the text box The Interval is used to specify the speed of the animation while navigating through an nDim The time that each frame is displayed should be entered into the text box by pressing the arrow buttons If the image displayed contains Event Markers these will also be reflected in the Image Navigator dialogue For the example shown three events are identified and labeled as Inject 1 Inject 2 and Inject 3 respectively By accessing the Go to Event dropdown list it is possible to move directly to the location of the nDim series associated with a particular Event label If the Navigator is positioned at an Event Marker the marker label is displayed above the dropdown list as shown below 88 Andor Technology Andor iQ USER GUIDE Time 0 00 04 339 himesms Frame Number Minrange Maxrange o Increment Interval e ms a Fo Bo o E fi ifam hi i m i e pobo i 1 cel a Set Min Set Mas Inject 1 1 Inject 1 Y ta SO 692 546 URIZ A xs see e iniect 2 3 Inject 3 The Jog Left and Right butt
398. nnel from the protocol The available keywords or Actions SE Flepeat Montage that can be used in a Protocol Tree are described Chapter 4 62 Montage Positions 4 2 by 2 J Ho 28 Positions 3 in some detail in 2 Repeat 9umin 11 planes 2 Snap Liye End Z Move To di End Montage Adjust Camera End XY Delta Z i End TO Acquire Edit Insert Delete Change Channel Andor Technology Andor iQ USER GUIDE 2 6 2 Analysis The Analysis Tab is used to calculate statistics for the current image or if drawn onto the image user defined regions Results are displayed in both graphical and numerical format Further details concerning the Analysis page are provided in Section 4 6 Graph Data Options Mean W Auto Range Show Event Labels Show Legend Print Mean Intensity To zoom in drag to bottom right To zaon Acquisition Analysis BGE Analysis Calibration Mowe Editar Spot Analysis Analyse EA Save amp Settings Data a Pegon ares muen Frame Tine tns Bet Mean ns 4093 26 m 1287 10 13 i 4055 20 1 J36 18 7213 10 14 E 4053 26 2 516 13 0174 10 37 dme O l 1 a O I W Automatic Directory Selection Group Analysis Greys Per Second Background Correction Ss Threshold i Clipping Intensity plots fr taco Mean Intensity Peak Intensity SE Area _ Area _ Integrated Intensity Zlntegrate
399. nology Andor iQ USER GUIDE 4 6 7 Multi Channel Z Stacks with Different frequency DIC It is common to acquire multi channel images at different rates for the channels Py Protocol Test Case JMC with Ret Ee Image Test Case 7 Z First i Protocol Type Time Seres F Camera Binning Use Current T walt Allocation Of Camera Selection Use Current Events dP Pause Click OF to start protocol gi Repeat T 10 times 2 mir Bee J Move Channel DIC al Snap gi Repeat T1 5 times O me fastest Repeat 10 um inii planes ia Repeat Channel Ch Ch Cha H Channels Description al Snap i End Channel End 2 i End 11 i End TO One example of this is in capturing a reference image while observing fluorescence dynamics such as signaling or translocation This example Protocol shows a DIC channel being acquired at a rate of one fifth that of the 3 channel fluorescent stacks 4 6 8 Multi field Multi Channel Z Stacks with Autofocus Protocol Test Case 10 T L MC MF Z Image Test Case 10 T L MC MF Z a Protocol Type Time Series ab Camera Binning Use Current Wait Allocation lf Camera Selection Use Current Events i AY Scan Test Compatibility Focus dP Pause Click OK to start protocol Ga Repeat T 60 times 5 min ai Auto Focus Activate Every 10 times RA Auto Focus D0 oa Repeat nr AT Positrons 3 ta Repeat Channel Ch Ch Cha H
400. nominator interrogated using various tools including through series analysis for characterization and 5 Background Mask Calibrati OS i e a quantification of intra and inter Numerator Background cellular dynamic processes Region Mean l Numerator Mone Threshold 0 Denominator Threshold O Denominator Background Region blean Ok Cancel Help Andor Technology 379 Andor iQ USER GUIDE The ratio tool is accessed by selecting the Process option from the iQ menu and clicking on the Ratio Image item Fie Image List Settings OC Wizards Process Analysis Plug Ins Help Enisodic Average Current lL O Mask Wizard es User Administrator Field Split Configuration Orca Com p Rotate Image Image Filter Image FITC Rolling Ball Filter Threshold Image ImageType 12 bit grey Clearview Deconvolution m Best Z i Image Size 1344 x 102 Image Math Rato Image Image Image Math Dual Image Disk Plug in List x C Dimension Editor C Flip Rotate Image Threshold Image Clearview Deconvolution Selector Views Orthogonal Slice View C Monadic Image Ratio Image C Math Image The use of the ratio tool is explained with the aid of the dual wavelength time series data of HEK cells mentioned previously and loaded with Fura2 the most widely used excitation ratio indicator for calcium The data set comprises two hundred ti
401. ns specify grey levels to Rectlinear Section f Default OpenGL Output Image Size Be xi specify render mode switch perspective and render crop in X Y Z Perspective Constant quality rendering a calibrated grid in the rendered image 307 Andor Technology Andor iQ USER GUIDE Animation Settings sill P E z Length 10 Time 0 l animation is specified by 10 f 15 38 rames Zoom 3 8 m a series of Key frames 10 frames 16 3 In Animation settings the Rotation About A6 which record AboutY 3 Set Position Visualization state when About Z you click in the Key __KeyFrame __Dekteal Frame button You can Add Visual References Advanced Rotation e m Animation finished exact values using the T Close Rotation Zoom Length set these parameters to and Time settings or you can interactively adjust for nice views and record those When you click on Animate the renderer settings are interpolated from one Key frame to the next and the interpolation rate is set by the Frame Length setting which can be varied between Key frames for maximum flexibility 5 11 2 Note on Graphics card and drivers Note Depending on Graphics card and drivers you may get some interaction between the OpenGL code and DirectX code which is used for the Main Image Window Examples of problems we have encountered are flashing or incorrect update of the DirectX w
402. ns These values are settings you must Grynkiewicz Poenie and Tsien equation determine for your own imaging setup for Ca transformation Ca Kd x IR Rmin Amas A 2 912 SBZ and are used during the calculation of i Calcium O Ca concentrations The actual values ame my for these parameters are set up in a Units dialog box like this one The Almers Kd 224 Ridin DA si 2 o E sho JE Neher method has a similar dialog box For the HEK cell example the following values have been established Rmin 0 41 Rmax 9 26 Sf2 36 Sb2 6 and Kd 224 nM The equation for the Almers and Neher method is expressed as follows Ca K R Rmin Rmax R The constants used during the calculation of Ca concentrations are as follows K 1819 Rmin 0 41 and Rmax 9 26 Selecting the None option does not apply any calibration to the ratiometric image a floating point image of the ratioed wavelength intensities will only be displayed the pixel intensities not expressed as values of actual ion concentration Once a transformation has been defined as described above the calculation of ionic concentrations is performed by clicking the OK button at the bottom of the Ratio panel Andor Technology Andor iQ USER GUIDE Ratio ND Image A floating point ratio image is generated for each time point in the temporal data series and the appropriate calibration applied to enable the conversion of
403. nsures that access times are optimized and provides the best performance with the large blocks of data created with multi dimensional images 16 Andor Technology Andor iQ USER GUIDE 1 6 Related Andor Systems Products We hope you are aware of the range of products offered by Andor Technology for quantitative fluorescence and luminescence imaging Having entered the field with iQ these other products might also help in your application of scientific imaging Further information can be seen on the Andor Web Pages at www andor com Full details will be sent on request e Imaris Andor acquired Bitplane AG the creator of Imaris in December 2009 Imaris is the leading 3D and 4D interactive image visualization and analysis Suite for life sciences Our teams are working together to bring optimum compatibility between our software products and will make announcements as progress is made In the immediate future please visit the Bitplane website for more information www bitplane com e KOMET enables you to image and analyze the samples produced using the Single Cell Gel Electrophoresis Comet Assay The Comet Assay is a technique allowing DNA damage in a single cell to be detected using a fluorescence microscope Live images from the microscope may be examined directly using a sensitive CCD camera or digital images may be imported from other sources Operations are initiated from a convenient toolbar and all data is stored in standa
404. nt Generic Shutter tab as the first in sequence It will start with all the settings showing Current Select the appropriate line by left click on the Shutter number and then right click and options will appear in a pop up menu as follows Andor Technology 577 Andor iQ How To Documents In the Pre Frame Action select the Shutter action required immediately before a frame is triggered In this case the Green Yellow Gr Yel source will be switched ON Then in the Post Frame Action select the Gr Yel Off action which will switch OFF the source immediately the frame has finished exposing Since the LLE uses negative logic and GS TTL control uses positive logic and defaults its outputs Low you will need to make sure that the other TTL outputs are set to OFF as shown to ensure that only Shutter 0 Red Of shutter 1 Grtrel On Fre Frame Action shutter 2 Cyan Ct Shutter 3 IMO Shutter 4 Teal Ott Shutter 5 Blue OR Shutter 6 Current Shutter 7 Grvel select y el Fost Frame Action Shutter O Red Ott Shutter 1 Grerel Of shutter 2 van Or Shutter 3 Uy Of Shutter 4 Teal Of Shutter 5 Blue O shutter 6 Current Gr Yel SE Current arrel Select Gr Grivel Select vel rel Select Grivel 1 rel Select Yelfor the Gr Yel Source is ON during the frame acquisition 10 Note that the Green and Yellow LLE channels requir
405. ntegrated Intensity the integrated intensity within the threshold limits specified in the Settings dialogue for each of the red green and blue channels Chromaticity color fraction analysis r R R G B i e the red chromaticity value equals the mean intensity of the red pixels divided by the mean intensity of the red pixels plus the mean intensity of the blue pixels plus the mean intensity of the green pixels g G R G B b B R G B Minimum Threshold the lower pixel intensity threshold limit as specified in the Settings dialogue for each of the red green and blue channels Pixels with an intensity outside the minimum and maximum threshold values are ignored Maximum Threshold the highest pixel intensity threshold limit as specified in the Settings dialogue for each of the red green and blue channels Pixels with an intensity outside the minimum and maximum threshold values are ignored Region defining coordinates the X and Y coordinates of the top left bottom and right points of the minimum enclosing rectangle of the region 257 Andor Technology Andor iQ USER GUIDE 2r U2 34 15 55 44 52 09 57 76 37 04 Delete EB Save The Delete button deletes the entries from the previous analysis from the Analysis Results table Once an analysis has been performed clicking on the Save button will open a save dialogue window allowing the user to specify a directory and file name to which to save th
406. ntensity v Mean Intensity Peak Intensity SD selected from the Dropdown a AACA listbox Integrated Intensity Zintegrated Intensity Integrated Intensity Per Unit Area The plot to be displayed is The available plots for display will depend upon the statistics selected on the Options page for calculation at the start of the Analysis SAS O Andor Technology Andor iQ USER GUIDE lf the Show Event Labels checkbox is enabled Event Markers and associated labels will be displayed on the Graph page lf the Show Legend checkbox is enabled the Legend will be displayed on the Graph page If a suitable printer is installed a hard copy of the charted data can be produced by clicking on the Print button FH The Colour Box button allows the colors of the Graph Background Labels and Grid Lines to be changed 5 3 2 The Data Page Acquisition Analysis AGE Analysis Calibration Movie Editor Spot Analysis The statistics Analyse EA Save Settings calculated depend Graph Data upon the statistics Class Area mutmu Frame Time ms Mean Intensity _ selected for wat calculation on the 12 AR E504 42 9716 2 B e24 4 12395 44 4762 2 Bn 5 915416 434715 start of the Analysis 2 Ba 6 18419 43 7907 wa Qe s214 7 22162 144843 Mean Intensity 112 246 3214 8 25372 43 9566 112 263214 9 28337 STH the Mean 2 e324 10 31341 45 2489 intensity value of the 2 Qess214 11 934362 46 6086
407. ntinuous shutter between the slider setting and zero output 494 Andor Technology Andor iQ USER GUIDE A3 12 Andor PZT X00 Piezo Stage control The Andor PZT X00 Z stage can be supplied in ranges 100 200 250 and 500 nm The 250 and 500 stages are required for multi well or micro plate operation having a large aperture to accept well plate holder accessories The PZT 100 shown and 200 nm stages are suitable for 35 mm Petri and MatTek dishes as well as 3 x1 slides Control of the PZT X00 stages are via a serial Curent z _ tive Read New RS232 interface and hence in the Device Acquisition Auxiliary Devices Setup Settings tab you will need to specify the Control Settings appropriate active COM port number for Status Connected On DEMO connection Once the connection is confirmed Device Select DEMO I Reconnect at Startup the device will show its status as seen left If the padlock at the bottom right of the dialog is clicked a warning will be shown that settings may affect device performance Accept the warning by clicking OK and then you will be able to set the Piezo Z range in the MICRON Range text box highlighted in blue This value is then used to scale the data set to the Piezo device controller and manage the internal Piezo Z values used for scanning and focus control 495 Andor Technology Andor iQ USER GUIDE Acquisition Auxiliary Devices Control is very
408. number of spots detected The current image is displayed in the Image Window and an overlay is Superimposed showing the identification number for each feature assigned during quantification and its corresponding bounding rectangle Multi Andor Technology 28 Andor iQ USER GUIDE 5 7 6 Reviewing and Editing Data Manage Analyse Review On completion of a Spot Analysis the results can be examined by clicking on the Review tab A panel is displayed as shown below Analysis AGB Analysis Calibration Movie Editor Spot Analysis Manage Analyse Review Data for up to eight parameter columns can be displayed for each feature and the Current analysis Colona Total number of spots 206 required parameter measurements can be Select Data to Show po M Area selected by ticking the respective check _Show Spots Vv Perimeter Show Images T Max Chord boxes as shown M Shape Clear Data M Integrated Intensity M Mean Intensity Save Data M Log Int Intensity M Mean Int Area 5 7 6 1 Show Data Table Pressing the Show Data button displays all the feature data calculated from a previous Spot Analysis in tabular form The quantified parameter data for each feature is listed as a row in the table listed in order of ascending number The numbers in the column Cell No correspond to those assigned during quantification A data table from the analysis of an individual image is shown
409. nutes When the wizard is running it shows you a progress dialogs as shown below for Registration and Background DSD Calibration Progress Status co Calibrating 115 Andor Technology Andor iQ USER GUIDE Background Correction Calibration Progress Status Calibrating OSD CF Model HiSec DSD Fite GFP T 1 The DSD calibration Wizard should be run each day before starting work the system will remind you if you do not do this The wizard should also be run whenever a filter turret is changed A filter turret change Wizard is also available from the Wizard menu or the DSD device control tab as shown below Note that Andor DSD turrets shown below can be switched with the DSD attached to the microscope 116 Andor Technology Andor iQ USER GUIDE DSD Filter Change Wizard DSD Device Step1 Step2 Step3 Step4 Step5 Step6 Finish Please Note You don t need to remove the OSD or camera fram your microscope to perform this operation Individual components are shown for clarity only Prepare to remove your filter turret as follows Lift the lid using finger qroove taking care notto lean on the camera Fig 14 Remove the 2 screws with 3mm Allen wrench Fig 2 Thread the screws into the open M3 holes leaving the heads high enough to comfortably grip with your fingers Fig 3 Remove the last screw and keep ttfor later Fig 4 Click Mest to continue Next Lancel Acquisitio
410. o camera suggest that the minimum Shutter Delay for such cameras is 50 ms A lower value will result in the first field of the current frame being acquired before the shutter has fully opened or closed This will result in you seeing an image containing horizontal lines Note If the Shutter State is set to Open Closed or Closed Open there will be one delay while the Shutter opens the image will be acquired and then there will be a second delay while the shutter closes If the Shutter State is on a single action i e Open or Closed then you will just see one delay Close Shutter On Move The Close Shutter On Move command can be selected deselected by clicking in the check box When selected the checkbox is labelled with a tick and after moving the wheel to a new filter position the shutter will automatically close Command Status 1 The command being written to Move Wheel 2 145 the COM Port is displayed in the Command Status section of the dialogue You should not send further commands to the filter wheel if a command is currently being written Press the Save button to save the current settings to file This will open a standard Windows save file dialogue from which you can specify the filename drive and directory to which to save the current settings The settings can be reloaded at a future date by pressing the Load button This opens a standard Windows open file dialogue from which you can select a
411. o column 696 560 Andor Technology Andor iQ How To Documents A Penrose mask is used for alignment To achieve alignment the camera must be shifted left right relative to the DSD 9 You can also check the focus of the Mask This is unlikely to need adjustment but if you are concerned about it please contact an Andor product support specialist for more information 10 Once the problem has been resolved you can reset the Debug Options 0 via the same Devices in file 11 If the system continues to fail in iQ alignment calibration then you can as a qualified Andor engineer run the land marking procedure detailed below 12 Land marking is required for consistent and accurate registration of adjacent conjugate calibration patterns created as an intermediate step by the DSD Revolution system 13 Once the system is mechanically aligned run an alignment calibration to completion Don t worry if the calibration fails just continue to the next step Andor Technology Andor iQ How To Documents 14 Navigate to C Program Files Andor Bioimaging Common Files DSDProcessing lmageu and double click ImageJ exe to open the application 15 From the ImageJ Plugins pull down menu choose Andor Landmark Calibration as shown below ImageJ File Edit View Favorites Tools Help o Back B P Search E Folders E Address C Program Files Andor Bioimaging Common Files DS5DProcessingiImage File and Folder Ta
412. o ease image matching and the T and R images of the pattern follow the same optical paths as the T and R images from a real specimen Consequently the pattern 114 Andor Technology Andor iQ USER GUIDE images are distorted in exactly the same manner The distortion is wavelength dependent and so the registration algorithm is applied at all filter positions and parameters stored so that they can be applied during image capture based on the filter combination in use K The DSD calibration wizard also J Recadl New Measures the background signal or Camera Andor AMH Andor DSD Andor APZ image for each wavelength with the Acquisition Ausiliary Devices DR328 1158 Settings DSD Processing light source in the correct state and W Enabled Catt snnnt filter position So the wizard corrects maata ee for optical distortion and background f Confocal Confocal amp Widetield pane EEES to provide the best quality result with eas the widest dynamic range Confocal Black Level O ss MEL The Wizard executes automatically SE a from a single button click on the Camera user interface as shown left Simply click on the Calibrate Alignment button highlighted in red Note you can select and deselect the Background Correction option by clicking on the blue highlighted checkbox The only reason to deselect is for reasons of speed as the process adds about 90 seconds to the Alignment step of about 2 mi
413. o software jitter and varying time for device movements this may mean that the timestamps for images are not uniform The recorded time is a truer acquisition measurement compared to a computed timestamp Return to Table Snap Stream Overlap ON OFF Acquire a series or burst of frames from the camera while the camera is set to Free Run Stream mode The sequence differs from Frame mode above in that frames are not triggered one by one but are taken as soon as they become available The camera is continuously acquiring and at the end of each exposure iQ will start to move devices if required during the camera readout period Hence synchronization will depend on the speed of the devices and is not guaranteed but is recommended for fast devices like Piezo Z Laser combiner AOTF or other fast light sources Slower devices may not be synchronized Devices which can stabilize within the camera readout period will ensure the highest performance If synchronization is unreliable then revert to Frame mode If Overlap is set to ON the camera will run in Overlap or Frame Transfer mode and achieve maximum frame rate Pre frame and post frame actions are executed so fast shutters can be used but may be unnecessary Note As the camera is running continuously in Stream mode the fire pulse will be generated continuously When using an ALC device with the fire pulse connected to the Active Blanking input this can have undesirable behavior Stream
414. ocessing 5 Memory manager SDK to allow third parties to integrate with Andor s powerful memory manager and gain benefits of ImageDisk technology 6 Andor Laser Combiner control SDK is also available and has been integrated with 3 party products including LabView and MatLab 7 Photonic Instruments photo stimulation products can be integrated into third party solutions and an SDK is available on request and agreed terms If you are interested please contact by email confocalsupport andor com 19 Andor Technology Andor iQ USER GUIDE 2 The User Interface 20 Andor Technology Andor iQ USER GUIDE 2 1 User Management and Logon Procedure Andor iQ has a 2 level logon procedure First is the Windows logon and second the iQ logon both are significant To setup iQ logon to Windows as a local or system Administrator because this gives you full access When you logon to iQ you select a configuration from a drop down list to initialize the software into the desired configuration Configurations are created by the Administrator using the Configuration Manager utility Section 2 3 1 The configurations specify which hardware is to be controlled and loads the appropriate software controllers or start iQ in the Analysis mode if no hardware is used The Configuration Manager allows the Administrator to control Global Settings for ImageDisk and Multi level User Access MUA When MUA is ON permissions are varie
415. ogy Andor iQ USER GUIDE 1 x Features can be modified at any time by double Basic colors clicking on the color box above the Delete button E OE EE A color palette will open from which you can select AAAA n unused color then pr OK Th lette will SSS Em 2 unused color then press e palette E NE O O close and the color will be updated in the color box BEES E nnnmunnmannn a Custom colors Define Custom Colors gt gt a To delete a feature select it in the list so that its name is displayed in the text box then select Delete You cannot delete the Background feature When you have added a new Class in this case Feature 1 it is added to the ROI list as shown e Paula drosophila PBC_VerticalFlip_Rotate180 16 Bit 642 0 107 x 1020 0 107 4D File Edit Image List Region View Fast MIP I amp i MUA Alel DOOS T ma ro A Background _Crop _Cycle _FRAPPA Feature 1 Edit Classifications selecting the Set Default option displays the panel illustrated below allowing the current ROI to be reassigned to a new class In the example shown the ROI class has been changed from Cycle to Feature 1 and the new class definition for the Current ROI is displayed in the Image Toolbar Any new ROI s subsequently drawn on the image will be classified using the new default class Load Save load and save the ROPs The ROI positions and classes are saved to disk from where they can be reloade
416. ogy Andor iQ USER GUIDE q gt Device Configuration Manager Register Unregister Plugin Components U IMPORTANT Ifyour device driver fails to register check that any Support Files and i nregister Done Drivers have been installed KI3DViewPlualn dil KIBest2Plugln dll KICropPlugln dll ga BAN KiLinePlugin di Aa KlMaskPlugin dil in or Bioimaging KlMathPlug n dll a Common Files KlMonadicPlugln dil CI ContigurationFile rchive KlOrth iewPlugln dll A Graphics KIOSplitPlugln dil 39 Images KlRatioPlugln dll KlRegionPlug n dll KlSelectorPlugln dll Kl ThresholdPlugln dil select the file to be registered unregistered by defining the correct file path in the right hand panel and highlighting the appropriate file name in the left hand panel The default folder for Plug In software modules is the Andor Bio imaging Common Files folder A2 1 9 About Andor Technology oe ANDOR Bioimaging Division were Andorig com wee kineticimaging com UK Office US Office 100 Southcenter Court The Stables Tel 44 115 906 1354 Suite 900B Pet del See DAS otrelley Hall ae Fax 1 919 467 0608 i Fax 44 115 906 1353 Morriswville per f Nottingham UK e mail info kinetic mail co uk e mail info us kinetic mail_co uk NGS GFE NC 27560 USA 442 Andor Technology Andor iQ USER GUIDE ANDOR iQ Appendix 3 Hardware Device Setup 443 Andor Technology Andor iQ USER GUIDE A3 1 H
417. oits solid state digital mirror technology DMD to create a digital illumination systems ideal for parallel multi region FRAP and PA 3 6 2 MicroPoint tunable galvo steered pulsed laser delivery MicroPoint is a pulsed laser delivery system with bio and industrial imaging applications It is available in manual and galvo steered versions and delivers 337 nm pulses from a Nitrogen pulsed laser 3ns 70 uJ via optical fiber to a dye 133 Andor Technology Andor iQ USER GUIDE cell resonator Dye cells are available to convert the UV pulses to more than 20 user selectable wavelengths ranging from 365 to 580 nm Custom designed achromatic microscope adapters ensure that near diffraction limited pulses are delivered to the specimen MicroPoint is an excellent photo stimulation tool providing ablation uncaging activation and bleaching capabilities adapted by tuning wavelength and energy Simultaneous and precise illumination and ablation of multiple regions of interest minimizes collateral sample damage Angular and spatial alignment of the illumination at the sample target is manually controlled via a 2 axis joy stick Focus in the z direction is manually controlled with a knurled focus ring 3 6 3 The FRAPPA Device Andor s FRAPPA uses a patented dual galvanometer scan head to provide a computer steered laser beam delivery system with no scan or f theta lens It can be configured in line with a CSU and or camera
418. ol In this example a multi channel time lapse protocol is shown with 3 channels To add a scan into this Protocol Tree select the Repeat T keyword in the Tree and then click the right mouse button and a pop up menu appears choose Insert The Insert Item dialog shown on the left appears and you choose an Action XY Scan in this case Next the Select Scan panel appears as shown over the page Andor Technology Andor iQ USER GUIDE Please selecta scan to use scan Setup WFxsF Mon negli MEXIF Mon macan summ MFx5F Test MF Compatibility Montage Test MF Mon Current Fi Test v2 Compatibility Calbratig z Scan Protocol Mewtest mai Image Test Case 1 Frappe Mult Phase Tim Protocol Type Time Series ah Camera Binning Use Current Wait Allocation Of Camera Selection Use Current Events fF AY Scan Test MF Compatibility 2s Focus dP Pause Click OF to start protocol pa Move Channel Ch Repeat T 20 times 200 mes Jo Repeat H ty Positions 8 Repeat Channel Ch Ch2 Ch3 End Channel W End i End TO Multi well Montage 13 000 14 000 15 000 4 16 000 17 000 18 000 19 000 20 000 2 21000 f lt n seen enn rr aa oa For a 23 000 sa oo 24 000 12 000 JFields Current Position 14 000 16 000 18 000 20 000 22 000 24 000 26 000 You can then select a Scan from
419. ol located in the Image Toolbar Section 2 10 2 selecting the Current Region option opens the panel shown below Blix VA Nele Doo T im eo Background _Crop Cycle FRAPPA _ This enables the current ROI displayed in the image to be Edit Classificationts reassigned to any class in the definition list Alternatively the current ROI can be reassigned to a new class created using the Edit Classification s function 80 Andor Technology Andor iQ USER GUIDE New classes can be created by clicking on the Add button or removed from the classification list using the Delete button Pressing the OK button accepts any modifications to the ROI class list Cancel exits from the classification editor ignoring any changes that have been made The Background Crop and Cycle features will be there by default and cannot be deleted These are always assigned the color Red Blue and White respectively Features must be assigned unique colors Add Delete Ok Cancel To add a feature click on the Add button and the following dialogue shown below will be displayed ioj x Enter a name for the new feature in the text box and press the OK button Following the definition Enter Phase Mame Feature of a feature name a color for the feature can be selected from the color menu shown below Clicking on the OK button will add the new feature to the Feature Definition list 81 Andor Technol
420. ology Andor iQ USER GUIDE Fields 32768 11852 261716 22451 959615 120 670000 Fields 32768 11378 261716 23158 959615 145 670000 Fields 32768 11852 261716 23158 959615 145 670000 Fields 32768 12326 261716 23158 959615 120 670000 Fields 32768 13877 475771 22389 277062 112 340000 Fields 32768 14851 475771 22389 277062 112 340000 Fields 32768 13877 475771 23096 277062 116 500000 Fields 32768 14851 475771 23096 277062 112 340000 Fields 32768 14825 475771 23096 277062 112 340000 Fields 32768 16376 689827 22326 594508 108 1 70000 Fields 32768 16850 689827 22326 594508 108 1 70000 Fields 32768 16376 689827 23033 594508 108 1 70000 Fields 32768 16850 689827 23033 594508 108 1 70000 Fields 32768 17324 689827 23033 594508 108 1 70000 Fields 32768 18875 803883 22263 911955 104 000000 Fields 32768 19349 903883 22263 911955 104 000000 Fields 32768 18875 803883 22970 911955 104 000000 Fields 32768 19349 903883 22970 911955 104 000000 514 Andor Technology Andor iQ USER GUIDE Fields 32768 19823 903883 22970 911955 104 000000 Fields 32768 21375 107939 22201 229402 137 330000 Fields 32768 21849 117939 22201 229402 129 790000 Fields 32768 21375 107939 22908 229402 137 330000 Fields 32768 21849 117939 22908 229402 137 330000 Fields 32768 22323 117939 22908 229402 137 330000 Region Info Fields End Environment User iQ User Configuration iMic Luca Environment End Protocol Description Protocol TL 3Ch MF Image TL
421. om the image list and press Ok 6 10 1 3 Dimension of Interest Wavelength Time Exposure Mey Or KI Dimension OF Interest Select the Dimension of interest from the dropdown list and then select the type of 374 Andor Technology Andor iQ USER GUIDE Range from the drop down list 6 10 2 The Background Tab Background f Use constant value of f Use maximum of area of interest number C Use image frame number along operating dimension C Use separate image Image me ET f Use maximum of area of interest Use mean of area of interest Background A Background B This tab is used to specify the Background Correction Values which will operate before the primary mathematical processing is started Background correction will subtract the value specified in the selection from each pixel in each image prior to the calculation Options included are e A constant value can be specified e Maximum or Mean of a numbered Region of interest e One frame from the Source Image s A B e A separate image in the image list can be used Select one of the options and type a value into the text box for both A and B if applicable If Use separate image is selected use the select button to choose an image from the image list 375 Andor Technology Andor iQ USER GUIDE 6 10 3 The Mask Tab Mask Options f Mo Mask Masked pixels set to value 0 CO Mask where atter background subtraction SOU
422. ometrics Princeton Instruments Targa Adobe TIFF standard Andor Multi TIFF UserDef allows Raw import of images with known bit depth XY and header size Visilog Zeiss LSM lsm Andor Solis SIF Images may be exported from iQ using any of the following formats AVI BioradMRC Bitmaps Fenestra JPEG jpg joeg Kontron Micro Voxel Photometrics TIFF files 2 4 1 Five TIFF formats are available for saving images TIFF multi image file Image saved in a multi frame format retaining its original bit depth TIFF 8 24 bit multi image file Image saved in a multi frame format Bit depth information is retained if the original image is 24 bit or 8 bit data For 12 bit and 16 bit depth images the image is subsequently saved as 8 bit data TIFF single image file Image saved in a single plane format retaining its original bit depth TIFF 8 24 bit single image file Image saved in a single plane format Bit depth information is retained if the original image is 24 bit or 8 bit data For 12 bit and 16 bit depth images the image is subsequently saved as 8 bit data TIFF LZW A compressed image saved as a multi frame format 40 Andor Technology Andor iQ USER GUIDE 2 5 The iQ Control Window The Control Window has three main parts gt Andor iQ 2 2 Andor Camera File Image List Settings Wizards Process Analysis Plug Ins Help e Toolbar Y a amp ry P Open Imaris i Current e Function Ta
423. on Origin 1st 0 4 Modify 0 4 Modify wavelength fhm 1 Modify zo o O 1 Modity Montage ffad 8 82 Modity O TE Modify Time m Modify NB To edit 1st and 2nd dimension use XY calibration Ok Cancel Help Dimensions may be added to the list by clicking on the Add button and completing the appropriate text boxes This function is especially useful for correcting corrupted image data e g where the Event dimensions may have been lost from an nDim series Modifications to the edit table may be cancelled by pressing the Reset button Any changes made to the Dimension Editor are permanent and irreversible and are accepted by pressing the OK button Pressing the Cancel button exits the edit panel without accepting any changes to the selected image 77 Andor Technology Andor iQ USER GUIDE 2 8 2 6 Selector The Selector is accessed from the Edit menu Selector ND range selection l j Name PluglnTest TIF Type 32 bit floating point greypseuda Intensity 1 000000 lus x 160 1 000000 urn y 120 1 000000 um Time 20 msec item brings Up the panel Clicking on the selected shown on the left Resultant Selector Details Dimension Dimension Hame Start Interval Units Time Min CT J 4 Interval adnan Set Navigator io Set Navigator Resultant Image Name Plugl AT est TIF Selector Lreate Ok The Selector enables images to be sub sampled along
424. on the camera body for Camara Alins FL maslo Camara physical identification in this case the SN is printed as X3877 Note is associated with Casi configuration FLZ master which we created in the previous section When you click on OK iQ starts and loads the camera in FLZ mode and connects to the PCU or BoB and any other hardware you specified We will configure this instance of iQ in the next section kamera oe eciion Now start another instance of iQ and select the TL Slave configuration When you do Please select an Andor camera po E another camera selection Camera Alias TL slave Camera 1 dialog is shown Select the second camera to be use for simultaneous acquisition Cancel You will now have two instances of iQ open and you screen will look something like this where we have Master on the left and Slave on the right One large 24 or greater or two medium size 22 high resolution monitor s suit this application best 223 Andor Technology Andor iQ USER GUIDE Andor 1Q 1 10 2 Andor Camera With Fast Zand Wa Camera 4 0UG97_BV 3877 DIX File Image Lat Settings OC Weard Process Andya Plgdne Heb F blet Payot F Curent Contest User Ishihara Lab Administrato Configuration FLZ master Image Focus qe ANDOR jQ 14 bit grey pseudo 512 x512 o Acquisition Analysis BGB Analpsis Calibration Movie Editor Spot Analy bt Ide fuso Auto Sa
425. ons gt Device Configuration Manager sie Edit Settings Tools About Exit view Configurations ard New Configuration Open Configuration Fdit Delete Current Configurations ccf E Selecting the Open Configuration item from the File menu displays the following page 437 Andor Technology Andor iQ USER GUIDE ES Kinetic Device Configuration Manager xj File Edit Settings Tools About Exit Manage Kinetic Configuration Archive r Current Configuration Rewind Delete Done J Make a Backup Configuration File each time the Configuration Manager is Closed Old Configurations Use the control key to multi select kiconfig 16 06 2003 11 54 26 1NI Created By Mark O Date Created 16 06 2003 11 54 15 Date Last Modified 16 06 2003 11 54 15 Acquisition Device Kinetic Dummy Camera Driver File KIDStdDummy ocx kiconfig 17 06 2003 11 56 03 INI The Configuration Manager automatically saves a backup version of the configuration file to an Archive folder every time it is closed This ensures that no configuration data is lost and allows previous configurations to be recovered should the current configuration file become corrupted or accidentally deleted A catalogue of archived configuration files can be found in the Old Configurations list on the Manage Configuration Archive as shown above The details of each configuratio
426. ons can now be controlled from the keyboard by holding down CRTL and using the Left Arrow and Right Arrow gt This keys the frames to be played back and forth Use the CTRL plus the Up Arrow 1 Down Arrow V to select the tabs in a multidimensional image 89 Andor Technology Andor iQ USER GUIDE 2 10 The Image Toolbar The Image Toolbar is only displayed when ToolBar is selected in the Image Window View Menu It is used to define and manipulate Regions Of Interest ROPs on the image and to magnify the image view The tool bar can be rearranged to fit the users needs by holding down the left mouse button and dragging to the preferred location Bw NVA NN Tel DoS T i ma O 2 10 1 The Hand Button If regi h fi h OR qajaja regions have been defined on the ae image using one of the region tools Auto Map the hand lets you select and move the region Ne Edit Right clicking on the image or a selected ROI will provide a popup menu Delete All Load Save Set Reference Frappa Device Frappa Channel Frappa All Regions 90 Andor Technology Andor iQ USER GUIDE Move Select Tool The Select Tool has its own menu When ae selecting Select Tool the available options are shown in the zoom Out picture on the left This is a shortcut for accessing the options w NA on the Image Window Tool Bar without recourse to the ToolBar Point itself Line Polyline Freehand Polyline Rectangle
427. onvolution process is shown in Figure 6 8 1 Andor Technology Andor iQ USER GUIDE Blurred Loop Image estimate Deconvolved Image Figure 6 8 1 A flowchart of the Meinel constrained iterative deconvolution algorithm implemented in ClearView 365 Andor Technology Andor iQ USER GUIDE 6 9 Best Z The Best Z is accessed from the Process menu and is used to select the best z slice from a z stack image to form a new image whose z Episodic Average Mask Wizard dimension has only one point the one selected by the user Field Split Flip Rotate Image Image Filter Rolling Ball Filter Threshold Image Best Z Z DEVELOPMENT TEST REPORTS A Cear tiew Decornvolution Best Z displaved in host application s viewer then click the Select 2 button This will record the selected Z position and will set the image to the nest point Image Math Ratio Image Image Image Math Dual Image Disk When all points are done you can click Create to generate the selected Best image then Show to display the new Image in viewer The major use of this tool is for the user Channel offsets 0 0 to select the best focused slice from a z stack Usually the source image is not only a z Time 0A stack but also a time series or even Select Z Clear All multi channel time series Click the channel to set the best Z by moving CI __ Show __ Cos through the time series Once satisfied
428. or Fura 2 Fura 2 is excited sequentially at wavelengths 340 and 380nm when in the presence of calcium ions Fura 2 shifts File Image List Settings OC Wizards Plug Ins Help setup Wizard Channels Channel Manager New Edit Copy Delete Close Channel Summaries Currant Channel CH1 Eng Channel Fura 340nrm GA Channel Fura 2 380nm its absorption spectrum changing the resulting emission intensities whose emission image ratio 340 380 is used to determine Ca concentration In this example a Monochromator is used as the excitation light source Use the Channel Manager to create 2 Channels Fura2 340 and Fura2 380 The channels are now available for incorporation into a New Protocol The example shows a channel summary for the two required channels The Fura 2 380nm channel was created as described previously Click on the Close button to exit the Channel Manager Create a new Protocol with steps previously explained in section 4 4 2 Insert the two Channels Fura 2 340nm and Fura 2 380nm Edit the Repeat and Interval 203 Andor Technology Andor iQ USER GUIDE settings to define number and frequency of image pairs to acquire Note that you can either create two Move Channel AND Snap actions or you can use a Repeat Channel action see below q Protocol Mult phase MLAT a Image Multi phase MAT a Protocol Type Time Series ah Camera Binning Use Current Wait A
429. or iQ USER GUIDE following coordinates Questions Suggestions Requests and Bug Reports to http Awww andor com contact_us support _request 11 Andor Technology Andor iQ USER GUIDE 1 3 Access to Software and Installation 1 3 1 Modularity and Timed Evaluation Settings Andor iQ is provided as a series of modules which are available for evaluation and can be downloaded from our FTP site For more information please contact your local representative or go to http AWwww andor com contact_us support request 1 3 2 Dongle and Licensing and Updates When you purchase iQ modules you will be supplied with a dongle key which plugs into the USB port The dongle provides licensing for the specific modules purchased The current components are Core FastLZ Tracker ClearView Multi field and Multi well Analysis modules Each Module has a specific dongle code and will be accessible only if you have that license If you wish to evaluate any of the iQ modules you can obtain an evaluation license file which will be setup for 30 days Andor Evaluation licenses are available by contacting support at http Awww andor com contact_us support request 1 3 3 Download version from FTP or Web We provide a downloadable version of the software which can be obtained with password protection from a specified Andor ftp or web site Once downloaded and installed the iQ software can be activated by the user sending the name of the
430. or photo switching bleaching activation and ablation The products can be configured with Andor and third party products and provide market leading performance and functionality Please contact us for more information or see the Photonic Instruments web site www photonic instruments com e Andor Custom Imaging Systems Andor will tailor make a system for quantitative fluorescence or luminescence imaging to meet your requirements Visit our website to find out more Solutions for calcium GFP applications super resolutions and more visit our website for more details Andor Systems Division has extensive development facilities and expertise in both fluorescence imaging hardware and software Custom solutions can be developed 18 Andor Technology Andor iQ USER GUIDE 1 7 Software Development Kits SDK If you have programming effort available and would like to investigate writing your own OCX for a camera or other instrument SDKs are available on request from Andor The following SDKs are available and include skeleton OCX and example code provided for Borland Builder and Microsoft Visual C 1 The new Python IDE is a fast way to extend iQ2 for your specific activities 2 Camera OCX SDK to allow integration of cameras with Andor iQ 3 Auxiliary Device OCX to allow integration of instruments with Andor iQ 4 Plug In dynamic link library DLL SDK to allow creation of specialist tools for image analysis and pr
431. or subtraction but in order to obtain high quality results the images must be very closely aligned e g 0 1 pixels The DSD Calibration Wizard is described in section 3 3 2 3 3 1 DSD and SD SIM Principle of operation The DSD is the first confocal instrument to successfully deliver high performance confocal imaging using a cost effective white light source rather than lasers The differential spinning disk DSD scanner can provide multifluorophore imaging from a single light source at a cost substantially lower than laser scanning instruments However unlike previous white light systems DSD performance is comparable to that of point scanning systems but with higher speed and lower photobleaching DSD is competitively priced and uses a patented optical detection principle known as aperture correlation to reject out of focus light Key benefits of the DSD scanner include frame rates up to 10 times those of most point scanning systems optical sectioning with objectives ranging from 10x to 100x operation with many fluorophores by selection of appropriate filters and as will be discussed acquisition of confocal and epifluorescence images simultaneously In a DSD system the spinning element comprises a single synthetic quartz disk supporting a thin layer of aluminum in which the structured illumination pattern SIP is created by photolithography The aluminum SIP has a 1 1 mark to space ratio half metal and half space which mean
432. or you can configure a dual excitation wheel configuration in 1 FITC which one Excitation wheel contains ND filters for 520 a intensity control and matching between channels If Shutter Open two or three filter wheels are selected Wheels A B CG and C will show individual tabs on the Ludl Interface When you right click on any of the circles representing the different filters located in the wheel a dialogue box will appear Enter the Name of the filter the Numeric Value the Units and a Description if desired The Name Numeric Value and Units should be written on the filter If you click in the colored region area under the OK and Cancel buttons a color dialogue box will allow you to select the appropriate color to represent the Filter We recommend you use colors that directly correlate with the excitation and emission colors when setting up Filter Wheel Colors 479 Andor Technology Andor iQ USER GUIDE IMPORTANT NOTE You must give each filter position a different name to avoid confusion as the controller uses these names to index the wheel Filter Humence Value Units M Description Press Next to continue or Cancel to exit A3 6 6 The Jumper Settings on the Ludi MAC 5000 If using the MAC 500 with Andor iQ you may need to adjust the jumper settings to the following addresses Address 1 X module Address 2 Y module Address 6 Z module The MAC 5000 uses a simple jumper schem
433. ordinates immediately Return to Table Move Ref Z Move the Reference Z devices stage or Objective to the coordinate specified in the Action parentheses The coordinates will be recorded from the Reference Z device which is selected in the Select XYZ Device under the Wizard menu If you switch between Z devices in a session e g between microscope and Piezo then you should Edit the Move Ref Z Actions to avoid out of range or risk of collision between the objective and stage Return to Table Move Scan Z Move the Scan Z devices stage or Objective to the coordinate specified in the Action parentheses The coordinates will be recorded from the 197 Andor Technology Andor iQ USER GUIDE Current Scan Z device which is selected in the Select XYZ Device under the Wizard menu If you switch between Scan Z devices in a session e g between microscope and Piezo then you should Edit the Move Scan Z Actions to avoid out of range or risk of collision between the objective and stage Return to Table On Line Chart Insert the on line graphing tool This tool will show a graphic which is updated on frame Snaps showing the mean in regions of interest drawn onto the iQ Image Window It can also show the mean of ratios of the regions when two or more channels are present in the Protocol Return to Table Pause Pause lets you enter text which will be shown on a Windows Message Box with an OK button Execution of the Protocol will Pau
434. ormal Master Slave operation Master drives a multi dimensional acquisition and the Slave is triggered to acquire frame for frame The user must make sure that the Slave is set to acquire the correct number of frames to match the total number in the Master image If you follow these steps all should work smoothly and you should gather well aligned high quality data for your studies 124 Andor Technology Andor iQ USER GUIDE 3 5 Stage Alignment stage Alignment should follow System Calibration and once completed you will be ready to start acquiring images with your scanning stage NB If you never want to acquire images from a micro plate multi well or a montage mosaic then you can skip the stage alignment step Acquisition Auxiliary Devices E _tastve x sron NN If you are using a camera which contains a Camera Prior DUGG BV 3771 Seting back illuminated chip e g iXon 897BV or Exposure General Device Ifo _ lt lt 888BV then you may have to flip the chip Temperature Shutter Speed Amplifier Image Orientation Flip first about the horizontal or vertical axis to r None r Vertical fe Horizontal correctly align the stage axes to the Rotation in Degrees second y g microscope The chip is reversed in the C AntiClockwise 90 housing to present the back of the CCD chip for illumination Thus when the camera is readout one of the axes is reversed or flipped Make sure
435. otocol Repeat T 7 time O ms fastest eo Move Channel Current A el Snap sections Ea Protocol QO Run oP Channel LA Y Scan Device Setup Loop Protocol i Display Image Show All _ Ee Save Ag a Options Help 153 Andor Technology and Edit the Protocol by changing editing Repeat T loop parameters Number and Interval Be sure to select the correct Calibration setting for the microscope objective in use During Calibration you can add an Optovar magnification setting You can check Optovar in or out with the checkbox see blue rectangle on left screen shot More sophisticated multi channel and multi dimensional sequences can be easily created Read on to find out more about Channels Scans and Protocols These Topics are covered in the following Andor iQ USER GUIDE 4 1 2 How to Acquire a Single Frame Image Acquisition Analysis Movie Editor Spot Analysis chi Che Status Ere DIC Cha Select the Acquisition tab To see a Live Image from your camera press the Live button which you will see toggles to Idle indicating that pressing again will stop live imaging The Status panel changes color to Cyan when live To acquire a one frame image press Snap This can be activated whether in Live or Idle mode If you click on the Channel button a pop up menu appears showing Channels available to the current user If you select a Ch
436. out This is the time the software will wait for a trigger event When this time expires the software will abort the image capture A3 2 2 2 2 The General Settings Tab The General Settings Tab allows the user to change selected features for your camera There are 3 additional tabs located under the General Settings Tab These include Temperature Shutter Speed Amplifiers and Image Orientation Note These settings are locked to prevent accidental changes If Multi Level Users is selected in the Global Settings of the Configuration Manager then only administrators or power users will be allowed to make changes to these functions 453 Andor Technology Andor iQ USER GUIDE To unlock or lock the settings press the following button The user will then be warned about the impact of unlocking settings Once changes have been made select the same button to lock the settings again A3 2 2 2 2 1 The Temperature Shutter Tab Exposure General Temperatures Shutter SpeedAmplifier Image Orientation Temperature and Far F0 100 a h 20 Actual Temperature 7 Read Cooling Working Fan i Full Low O Click to Lock or Unlock gt gt gt The Temperature Tab allows the user to adjust the temperature to a specific temperature by moving the scroll bar The selected temperature will show above the scroll bar The actual temperate and cooling status will also be displayed und
437. ove and redraw lines for analysis All you have to do is click on OK when the error message shows and the Kymograph Plugin will recover and continue This will be fixed in a future release 318 Andor Technology Andor iQ USER GUIDE Reference Hinz B Alt W Johnen C Herzog V Kaiser H W Quantifying Lamella Dynamics of Cultured Cells by SACED a New Computer Assisted Motion Analysis Experimental Cell Research 251 234 243 1999 Article ID excr 1999 4541 available online at http www idealibrary com 319 Andor Technology Andor iQ USER GUIDE 5 9 Orthogonal Slice View Orthogonal Slice Viewer C IMAGES ANTHER_8BIT_CROP TIF Single Slice M X The Orthogonal Slice View Window is opened when Orthogonal Slice View is selected in the Image Window Tools Menu Once opened the Orthogonal Slice View Window will display three orthogonal views of a selected point on the current image The main part of the image displays the current XY section For a multi dimensional Image containing a third dimension e g Z moving the crosshairs to a point on the image will display a ZY slice to the right of the image and a XZ slice below the image The coordinates of the position of the crosshairs are indicated in a box at the bottom of the viewer The visibility of the XZ and ZY slices is controlled by checking or unchecking the corresponding boxes at the top of the viewer The view can be mapped by clicking th
438. ows you to define a This should be of the form 1 3 4 7 12 30 i i Pulsar tSeiceuan list of wells which either have not been C Include Selected Items a used or contain no viable specimens Reject Selected Items The list is created by typing the numbers of the wells or ranges and you can choose whether this is the Rejection or Inclusion list with the radio button 172 Andor Technology Andor iQ USER GUIDE In the next stage you will define the fields to be imaged in each of the wells Two options exist one for Systematic Sampling and the other for User defined fields 4 3 4 1 Systematically Sampled Fields Fie ImageLst Settings QC Wizards Pugins Hep From the Calibration dropdown select the XY Setup Wizard Randomized meander Calibration that you will use for the acquisition Select Number of Fields gt iecti RS Khoo and ensure that the correct objective is A Cad selected Z Bak News gt corce_ Specify the number of fields to sample within Sampling Setup each well The arrows change the value by 1 Please select calibration FSC the 1 0 buttons change the value by 10 Please selectthe number of fields required A uniform random grid will be thrown into within each well specimen space in which the X and Y Number of Fields per ell 1 repetitions are at a uniform distance and the sampling Factor Number 2 94E 04 Sampling Factor Area Length 2 94E 04 grid is positione
439. path can be defined by a user with administrative rights and it is possible to save data to a different path using the Set Save Path option in the iQ File menu or specifying a different file path at start up Group Analysis Results are calculated for all regions on the image that are in the same group i e all regions of the same color as opposed to being calculated for each individual region A region can be assigned to a group by selecting the Pointer tool in the Image Toolbar right clicking on the region selecting Classify and assigning a color Greys Per Second This option can be used to extend the dynamic range to obtain quantitative information and to directly compare data The intensity information is calculated from the grey scale content and the result is normalized divided by the exposure time at which the image was acquired Hence the resultant values are expressed as greys per second and will reflect relative intensities over a greatly enhanced range This function should be used with background correction for best results Background Correction The background value is computed from the Group of Red regions defined on the image using the ROI Tools on the Image Toolbar If you select the Background Correction option then the Red region mean value will be subtracted from each pixel during analysis Threshold This option must be ticked in order to access the Settings button Statistics available for selection are Mea
440. pdown box button Binning can be set in the protocol with keyword Binning This avoids the need to have different channels for each binning This also means that binning in all channels can be adjusted in one setting Changing Binning will affect the size of the Live image and images resulting from Subsequent snaps and protocols while still displaying the same field of view In the above example the binning has been set to 1x1 If the binning was changed to 2x2 this would result in an output image containing 1 4 the number of pixels Binning results in a sensitivity increase of M x N where M and N are the X and Y Binning respectively The charge from an M x N rectangular neighborhood is integrated into a single value at the sensor output image Smaller output images speed data transfer times so acquisition speed also benefits 450 Andor Technology Andor iQ USER GUIDE Temperature and Display fps This parameter allows you to view a Live update of the peas Displavttos current temperature by checking the Live Update Actual 48 a S jv Live Update Checkbox The Temperature setting is changed in the General Settings Tab under the Temperature Shutter Tab Section A2 2 2 2 2 1 The Display frames per second can be changed by using the up down arrows or typing ina value This allows you to choose the Display updated frequency Gains Pre Amplified Gain Control Gains Pre Amplified Gain Pre Amp Gain
441. played in a progress bar as the image files in the directory are constructed into an n Dimensional image The default 3rd dimension is time but this can be edited using the Dimension Editor as described in Section 5 2 5 Only files with matching names are imported using the Multi Load option e g If two image series red01 tif red80 tif and red_01 tif red_80 tif are resident in the same directory red01 tif red80 tif image series If the red_01 tif image is selected the red_01 tif E Cathy2 confocal tst e Cathy2 confocal tO00O z0001 Hif Cathy _ confocal FOo00 20002 tif Cathy confocal t0000 20003 tif Cathy confocal t0000 20004 tif E Cathy _ confocal t0000 20005 tif Cathy _ confocal t0000 20006 tif i Cathy _ confocal t0000 20007 tif Cathy _ confocal t0000 20006 tif i Cathy2_confocal t0000 _20009 tif Cathy _ confocal t0000 20010 tif im Cathy confocal FOO00 20011 tif i Cathy _ confocal FOo00 20012 tif i Cathy2_confocal_bOO00_20013 tif i File name Files of type red_80 tif series will be loaded Save Image save an image to disk This opens a standard Save File dialogue in which you can specify a filename and directory to which to save the image When saving files the user should be aware of the fact that some file types support multi dimensional images while others support on single XY frames The main types handled by each format are listed below
442. r options Region Highlight Image and All Edit Image List Region View Open Image F Save Image P SEE Wavelength Time ChO 340 nm rame Number Minrange Maxrange Increment Interval s ms a gt fo f E lt foco 4 at alial moi mpm cpl am _SetMin Set Max o amp x 7 Automap 100 306 4 319 Ix e w 3 Save Display BMP F Regions Save Display JPEG P Highlight Print Image Al Exit 3 To save only the region outlines choose Region The resulting image in this case would look like this 4 To save the highlight produced for example by the Threshold option in through series analysis choose Highlight An example of this is 547 Andor Technology Andor iQ How To Documents 5 To save only the image itself choose the Image option 6 To save image plus ROIs and Highlights seen in the Image Window choose All 7 Both JPG and BMP options will show a File Browser window where the destination drive and folder for the image are selected and a name for the destination image can be enetered 8 If you selected JPG a JPEG Compression Quality window willl be displayed The default value is 75 giving a good compromise between image quality and file size A smaller value will decrease both the image quality and the file size 548 Andor Technology Andor iQ How To Documents A5 14 How to Copy Images to the Clipboard 1 To copy
443. r shown above there are three tabs as Time Wavelength and Z These are the image s dimensionality tabs whose number and name are determined by the displayed image Selecting e g Time tab will display the information about this dimension such as the number of frames along the dimension and their respective time points It also specifies the animation to be carried out along this dimension If the selected image dimension corresponds to Wavelength this has an effect on the behavior of the Multi Channel view function and hence the features displayed in the Image Window The relationship between the Image Navigator and the Multi Channel view is discussed in Section 3 4 4 The nDim animation buttons are used to animate through the frames of an nDim W The First Frame button animates backwards through the nDim to the first frame W The Play Backwards button animates backwards through the nDim one frame at atime 86 Andor Technology Andor iQ USER GUIDE I The Jog Back button is used to rewind the animation back one frame The Stop button stops the animation eI The Jog Forward button is used to advance the animation forward one frame The Play button animates the nDim displaying the changing x y dimensions for each time frame HS While the nDim is animating the Play button label changes Clicking on the button will Pause the animation and change the button label back to Play The Final Frame butto
444. ra Selection iXon 887 At this stage if there are no other cameras in use then you simply E Present TESE specify whether you have an Optovar in M In Light Path fi the system optional additonal mC Mount z Magnification magnification lens following the objective j g J This is mounted on a slider and can be Camera AH Pixel Size a pushed in and out of the optical path iQ 987 1 um vi provides a check box to specify whether Edicomeratist in use or not You also define here if you have a C mount adapter with a magnification different from 1X You can add cameras to the by wa SS Ore a N LINO ar di ete ton clicking on Edit Camera List button p Camera Definition gt T ixon 887 897 16 DU 888 DU 934 13 ixon 860 24 a Name description in the Alias box piss a ance _ Hamamatsu Orca AG ER 6 45 Camera Hamamatsu C9100 01 7 4 kamamatsu C9100 02 8 FA k aji Hamamatsu C9100 11 12 16 Hamapfatsu Orca I 6 7 amamatsu 4880 9 9 Cascade 650 7 45 f f Cascade 512B 16 to name the calibration setting in Cascade 1K 8 L _ CoolSNAP HO 6 45 conjunction with the magniification of oo i PCO Sensicam QE 6 45 the objectives chosen When you click PCO Dicam 9 9 COHU 4920 8 6 A A Qimaging Retiga EX 6 45 on the down arrow box highlitghted Ri E A Qimagin
445. ranges will change W together 2 Image Contrast Options View E 4291 000 4 Max E 1067 000 Min E 000000 Step Invert Tl Exclude The horizontal scaling of the histogram is linear which may make it difficult to study Gamma Coefficient Log Scale Histogram Map scales the histogram X axis v Zoom to Map Link Channels E 750000 Max E 1600 000 Min 1 000000 Step u histogram details in images with a low view dynamic range Selecting Zoom to around the Min Max values as shown opposite The vertical scaling is also linear by default so that low frequency grey values may be difficult to see on the graphic Selecting the Log checkbox transforms the histogram Y axis to a logarithmic scale emphasizing the low frequencies as shown opposite This situation occurs in fluorescence imaging where the image histogram may be dominated by high frequencies of background 69 Andor Technology E 1 000000 Step Ms 8 f1 000000 4 step Cancel Andor iQ USER GUIDE 2 1 3 Auto Mapping Tools i Automatic mapping can be EMNE Options View used single shot by clicking on this Gawma Invert Exclude button or checked on a Right mouse Gamma Coefficient popup in the Image Window or lower Gamma j Image toolbar The Automap function C e Exclude estimates the Mapping range values Sammacosticint c
446. ration wizard then selecting Move To allows the image feature under the mouse to be brought to the center of the field of view Thus when the user goes Live the feature will be visible Set Reference allows the user to specify a different location on the screen to move the feature on Move To An example where this is useful is with a fixed laser beam system where features of interest are brought to the beam location for photo stimulation FRAPPA Device selects which device should be active for either interactive FRAPPA or Protocol FRAPPA actions FRAPPA Channel specifies the Current FRAPPA Channel not that FRAPPA Channels are specified in the Select FRAPPA Wizard as shown below FRAPPA All Regions allows you to choose modes between FRAPPA the currently 92 Andor Technology Andor iQ USER GUIDE Configure FRAPPA FRAPRA Channels Activate EOS Remove gt gt Remove All gt gt Reset FRAPRA Device FRAPPA FRAPPA 2 10 2 The Pointer Button vik Qjajq If regions have been defined on the image using one of the region tools the Pointer button lets you drag resize delete copy paste and classify regions Move the mouse over the image When the cursor passes over a user defined region it changes to a hand Holding down the left mouse button and moving the mouse will drag the region to a new position Release the mouse button to drop the region When the hand icon is at a corner or the middle of
447. rces and here we focus on the metal halide variety A serial interface is used to control both Prior LumenPro 200 which contains Acquisition Auxiliary Devices Curent l N Live n Snap J Record New r Control Settings Status Connected On DEMO Device Select DEMO WV Reconnect at Startup Reset Curent n Live n Snap Record New r Camera Andor AMH AndorDSD Prior Lumen Andor APZ Control Settings AMH Filter Wheel 4 Intensity KI Shutter Shutter Open a 200 Watt DC stabilized arc lamp 6 position filter wheel and pseudo continuous shutter 0 100 1 increment The GUI for this device is shown below where its demo mode capability is shown If you have a real device connected you must specify an available Device ID in the drop down list named Device Select A USB connection is required The primary controls for the light source are shown on the Control tab of the Device Setup dialog The combo box is similar to the DSD filter selection but in this case only controls a single filter wheel which has a switching time of 60 ms between adjacent positions A right mouse action on the combo box allows you to edit the current filter information such as excitation wavelength While the slider the continuous grey scale annotation allows control of the output intensity setting with 1 increments shown at 55 here The device Shutter checkbox switches the co
448. rd spreadsheet format for plotting and statistical analysis Excel Macros are provided to automatically manipulate and view the data e Revolution XD laser spinning disk confocal systems Andor is a global distributor of the Yokogawa CSUX dual spinning disk confocal scanner We integrate the CSUX with Andor and third party manufactured products to create industry leading live cell confocal microscopy systems Our systems provide ultimate sensitivity with extreme flexibility offering confocal TIRF and FRAPPA and ablation laser microscopy solutions Visit our website to discover more www andor com microscopy e Revolution DSD spinning disk structured illumination systems Andor 17 O Andor Technology Andor iQ USER GUIDE co exclusively distributes and creates solutions built around the Aurox spinning disk confocal scanner which is based on the principal of aperture correlation Unlike other white light confocal instruments DSD provides strong optical sectioning even at low magnifications because out of focus light is sensed via a second optical channel and actively rejected Laser free operation makes the system highly cost effective and provides freedom in choice of excitation and emission filters Visit our website to find out more e Photonic Instruments Photo Stimulation Systems Andor acquired Photonic Instruments in April 2010 the Photonic products include Mosaic and Micropoint systems which can be used f
449. rds features lying on the boundaries of the image as their measurement would lead to an underestimation of true feature dimension After selecting which filters to use and specifying their Min and Max values click on the Next button to continue The creation of a new experimental protocol file is now complete The example shown is for a single image An identical dialogue is observed for a multi TIFF time series or a time series generated using third party software 284 Andor Technology Andor iQ USER GUIDE 5 7 4 2 Editing Protocols An existing protocol can be amended by pressing the Edit button which reveals the panel over File Image List Process Function Wizards Plug Ins Help Setup Wizard To edit an existing protocol select the Spot Analysis appropriate file name and image Set up analysis Press Next to continue Cancel Subsequent threshold and filter dialogues are identical to those described previously Select image to use in setup amp Selectfrom Image List Grabbed6 Grabbed5 Focus CA TEMPACOLONAIA TIF CATEMPACOLONAT PIC_Colona CATEMPACOLONAT PIC CATEMPACOLONAAYVI Colona CATEMPACOLONA AY Multi Load CAOPTILAS SPOT SEQUENCE 8BIT TIME_SHUT CAKINETIC TEST 215 TIF test200 TIF Radams16 tif Radams16 tif Old 3channel 3D View Grabbed4 Grabbed3 Grabbed2 imageNamel2 Grabbed Andor Technology 285
450. reation and we then move to setting up the calibration phase if it is required 389 Andor Technology Andor iQ USER GUIDE 6 11 6 Calibration lojx Clicking on the Calibration tab reveals Sas Takeda Calibration the panel opposite Following ratio Equation for Ca transformation background correction and masking a Almers and Neher Options calibration can be applied to the floating HGikiswic Poors ands _ Options polit rato image t0 convert Mamada pixel intensities to values of ionic concentration Two different calibration procedures are Source Background Mask Calibration Nurnerator Background Region Mas 1 Denominator Background Region Mas 1 available the Almers and Neher method or the Grynkiewicz Poenie and Grynkiewicz Poenie and Tsien GPT transformation Either may Tsien be selected using the appropriate radio button The GPT transformation is expressed as Ok Cancel Help follows alas where R is the ratio recorded under appropriate physiological conditions Rmin is the ratio recorded at zero external Ca Rmax is the ratio recorded in excess external Ca Sf2 Sb2 is the ratio of the signal at 380nm Sf2 in zero Ca to that in the presence of excess Ca Sb2 Kd is the apparent dissociation constant for the indicator usually taken as 224 nM for Fura 2 390 Andor Technology Andor iQ USER GUIDE GPT Optio
451. remove it to make any adjustments to the fiber focus position Finally perform a series of FRAPPA tests within iQ making sure to test points and ROIS 3 6 6 MicroPoint Calibration MicroPoint must be installed and aligned by a fully trained Engineer 1 Preliminary setup requirements A we assume that you have an iQ Configuration that includes all the devices you wish to use including the ALC with MPU selected camera and CSU B Make sure that you have a suitable sample for photo bleaching either Andor Test Slide 001 or a fixed Molecular Probes specimen e g Cells 2 where microtubules are labeled green excited at 488 nm C If the MicroPoint is being used for ablation use the fist surface mirror provided with the device to properly calibrate the system The MicroPoint ablates a series of holes in the mirror that are illuminated in transmitted light and used for calibration D Start Andor iQ 2 0 or later exe dated July 28 10 or later d Make sure that MicroPoint is connected on a suitable COM Port Note the FRAPPA calibration is independent of objective magnification but depends critically on the camera orientation alignment and pixel organization You may wish to create a Frap channel for MicroPoint as with the FRAPPA but this is not essential as the MicroPoint device is not in the light path and you may use the current channel settings to Calibrate 2 Run the FRAPPA Calibration Wizard from the Wizards men
452. rent Protocol Time Lapse EF Protocol Time Lapse 4 Protocol MO 4 Protocol MCZ Z First 4 Protocol MCZ 4 Protocol MultiPhase 4 Protocol MultiPhasevithFrappa 9 Protocol MultiRate 4 Protocol Ratio 216 Andor Technology Andor iQ USER GUIDE 4 6 12 Drag and Drop Capability In addition to the templates drag and drop capability has been added to the protocol tree Left click on a tree item hold down the mouse button and begin to drag 1 9 Repeat T 10 times O ms fastest 7 Release the mouse to insert the item Mi Casini Aes 0 ms fastest at anew location There are protocol Snap Frame er rules that restrict which items can be rn p dragged and the location that items may be dropped When an item is restricted you will see a restriction icon Here an attempt was made to drag the repeat inside itself which is an invalid action When an item is allowed to be dropped you will see the following icon Left Here the end is being dragged within the repeat loop this is a valid action Resulting in the Snap being removed from the repeat loop Right Gail Repeat T 10 times 0 ms fastest Q Repeat T 10 times O ms fastest ao Move Channel Current Move Channel Current 2 dy sees a Snap Frame Repeat Loops may be dragged All items within the repeat and end will remain intact A repeat may not be dragge
453. requires that the image has a Montage dimension iQ produces this dimension when it performs a Montage scan or a Multi field or Multi well scan with Montage enabled Such an image is shown in the iQ Image window below Note that the image also contains Time XY also Known as Field Z and wavelength dimensions 330 Andor Technology Andor iQ USER GUIDE R Multi a v 4 gt Time XY Montage Z Wavelength 4 0 Frame Number Minrange Maxrange Increment Interval s ms a af u amh fo 5 lt foon a alial mi e MEZES SetMin Set Max 147 216 1201 1188 x e x50 2 500 al To produce a Montage from the image above we simply select the View menu and then click on Montage and the Montage dialog will show The figure below shows this and the Options dialog has been overlaid for convenience Montage View C DOCUMENTS AND SETTINGS M BROWNEWESKTOPUQ IMAGES MFMONZT2CH E BX i fi E Sampling Factor Y Update Each Frame Create Montage Show Cancel elf Options Montage Options Seles m Blending C None DESKTOPMO IMAGES MFMONZT 2CH 2X2 16102009 TIF Brightest Pixel Matching Enabled Stage Precision um rms fi X Edit 1 0 faidi To Match E l M Current Image 10 0 RS Pixel matching has already been applied to the current image press recalculate to allow image to be reregistered on next rendering Recalculate Ok Cancel
454. rigger Out Wait Allocation XY Scan Andor iQ USER GUIDE 4 4 6 Actions in more detail Autofocus Execute an Autofocus action based on the currently selected device This will typically be a microscope hardware autofocus action e g PFS ZDC DF Note that the Autofocus device MUST be already initialized for this to function correctly iQ will warn you if the Autofocus is not initialized when you try to Run the Protocol The location of this Action within Repeat XY Montage and Well will define whether Autofocus is executed for these fields Note also that Autofocus Activate Action below must be in the Tree inside a Repeat Time Action Return to Table Autofocus Activate Specifies Autofocus Action periodcitiy So if this Action takes the value 2 the Autofocus Action will be executed on every 2 Time loop or if the value is 3 it willl be executed on every 3 Time loop This Action MUST be inserted at a Repeat Time Action Return to Table Camera Binning Edit this item with Use Current and Adjust Current In the Use Current case the Protocol uses the value of binning set in the Camera page of the Device Setup Dialog Alternatively the binning value can be forced by using the Adjust Cuurent setting When you select Adjust Current you will be directed to the Camera control page within the Channel wizard and the value you set here will be used within the Protocol Note that the exposure stting is automatically adjusted
455. riven from TTL then the LED device is on or off LED Driver LEDD1A 4 STANDBY Cw PULSE A3 8 1 TTL Connections and iQ control In order to control it for bright field imaging with FLZ you connect a selected TTL line on the PCU to the MOD IN input shown to the left Note the LED illuminator connects to the LED connector 485 Andor Technology Andor iQ USER GUIDE If you are using FLZ then Burst And Bright Field Control KE the TTL output DO4 is used Burst Control Bright Field Imaging Control for Bright Field shutter Burst B iv Enable control so use this line You Interval 20 fs xl a can specify the exposure Poser Stet eet ee i mej time on time for the LED in l C At Z Plane Bo increments of 0 1 ms in FLZ IF Enable ITT Copy To All Planes 7 or in 1ms increments in L 488 0 MPUIFRAPPA i Shutter Control D04 Open Time Lapse The FLZ Burst H csid MoveTo and Bright Field control is shown in the dialog below lf you are controlling the TTL line for Neto _ Earcel the Trigger controller then you will setup Pre and Post frame actions on the chosen line Pre Frame Action Trigger Out O MPUDO0 Low Trigger Out 1 MPUDO1 Low Trigger Out 2 Current Trigger Out 3 Current Trigger Out 4 Current nae aon See left for example of using the Trigger Out 6 Current CPUa Trigger device in Time Lapse on TTL Post
456. rofiey Yp 154 Red gt J 4 Green X The above picture shows a Zoomed Image with X Line Profiles and Mapping Tool ai This button opens the Mapping Scaling dialogue r ss When the Arrow button the second Field Split Window is pressed down X you can use the mouse to drag and drop one of the ROIs to the place you want tn the field je The Zoom button enables you to magnify the image displayed in the Image Window enabling you to view the fine detail of features of interest more clearly 349 Andor Technology Andor iQ USER GUIDE You can select one of the two radio a gt ROT ADI av buttons on top of the Field Split window named ROI1 and ROI2 and then click the Up Down Left Right buttons to move one of the ROls one pixel up down left or right Alignment Before closing the Field Split window you can save the Load Save alignment as an alignment file by clicking the Save button on top of the Field Split window You can also load alignment files into the Field Split window by clicking Load button at any point of the selection process Biase If you do not want to have the new image you can just close the Field Split window at any time by clicking the Close button This returns the Image Window to the original image You can change the width or the height of the ROIs by typing new Width 236 1 15 x2 28 values into the Edit Box at bottom Heigrt
457. rticular correction method to be applied is made by selecting the appropriate radio buttons Set Background to O or Ignore pixels below background The benefits of the Photobleaching Compensation algorithm are illustrated below Source Image User defined ROI Background ROI Photobleaching Compensation Frame 25 Definition of Compensation parameters Processing will be performed on the current Image Resultant Image Name 415 Andor Technology Andor iQ USER GUIDE Results of Through Series Analysis within the user defined ROI Prior to Compensation After Compensation 416 Andor Technology Andor iQ USER GUIDE 2 rEVHNOLUGY Appendix 1 Installing the Software 417 Andor Technology Andor iQ USER GUIDE A1 1 Installation of Sentinel Drivers If you are running Windows NT or above you will need to install Sentinel drivers which are required for your Software Protection Key Dongle without which your Andor software will only run in Demo Mode Note It is not necessary to install Sentinel drivers if running in Windows 95 or 98 Demo mode has all the functionality of the full version of the software except You cannot acquire images You can only open Demo images from your Andor Installation CD Rom You cannot save data To install the Sentinel drivers insert the CD into the CD Rom drive then run the program START EXE which is in the root directory of the CD This will start the Install
458. rwise the hardware settings will be inconsistent and iQ will reject the Protocol and show an error message iXon CSU FLZ Prior File Edit View Favorites Tools Help Back 7 pi Search Wey Folders Address C Documents and Settings All Users Application Data Kinetic Imaging iQ ixon CSU FLZ Prior BFR Folders e My Documents 4 My Computer S Local Disk C1 E E Andor E Documents and Settings ES ABD 3 5 All Users E ES Application Data 53 Kinetic Imaging E ES Common 4 Configuration 3 6 ie 3 FastLz 3 Ixon AOTF Test 9 iXon csu 3 ixon mono No Grab B Test 3 Test Ixon Ludl 3 Test iXon Lud DG4 L Microsoft 3 Desktop 3 Favorites E Shared Documents x A Name E 4d chartOptions _ Ei 4d KI_EXP E 4d KI_MERGE E 4d KI_NODE E 4d KI RATIO ka cec ChartOptions E cec KI_EXP E cec KI_MERGE E cec KI_NODE E cec KI_RATIO E cje ChartOptions 3 cjc KI_EXP E cjc KI_MERGE E cic KI_NODE E cic KI_RATIO E CSU_green ChartOptions ES CSU_green KI_EXP 3 CSU_green KI_MERGE S CSU_green KI_NODE E CSU_green KI_RATIO E DefaultPhaseList Ist E Events E Fast50 ChartOptions E Fast50 KI_EXP E Fast50 KI_MERGE Type CHARTOPTIONS File KI_EXP File KI_MERGE File KI_NODE File KI_RATIO File CHARTOPTIONS File KI_EXP File KI_MERGE File KI_NODE File KI_RATIO File CHARTOPTIONS File KI_E
459. ry mask to be created which can be applied to any image using standard mathematical operators prior to subsequent quantification The mask image enables features of interest to be isolated from unwanted noise in single plane or multi dimensional series and also provides a source of data for binary image analysis Note that this tool will perform the thresholds set for each channel through an entire multidimensional data set ami The Grey Max UE Ce Ej Min 381 menu m J gt Jv Multi Ok Cancel Help Panell selected item Episodic Average Mask Wizard Field Split Flip Rotate Image Image Filter Rolling Ball Filter Threshold Image Clearview Deconvolution Best Z Image Math Ratio Image Image Image Math Dual Image Disk Threshold Tool is accessed from the Process Clicking on the launches the set up wizard shown below A user defined threshold is applied to the source image to create a binary mask The functionality of this module is identical to that discussed in the section describing the use of the Ratio Tool Further details concerning the use of the Mask Tool can be found in Section 6 11 363 Andor Technology Andor iQ USER GUIDE 6 8 ClearView Deconvolution The ClearView deconvolution program is a module which will function with all Andor products including Andor iQ Komet Komet and future products It is accessed from the Process Menu Cle
460. s CATEMP SPOTFILTEREXAMPLE TIF_Thresholded Binary image Lower Threshold 20 Upper Threshold 255 F Multi Once you have set the Threshold Limits click on the Next button to continue with the protocol set up 281 Andor Technology Andor iQ USER GUIDE Pressing the Cancel button will close the Manage dialogue ignoring any changes to the protocol file Pressing the Next button moves to the filters section of the Wizard where filter limits are specified for the opened image The filters panel shown below is used to select which filters are to be applied to an image for subsequent processing and to specify individual filter parameters File Image List Process Function Wizards Plug Ins Help Setup Wizard Available filters are Area Shape Factor Spot Analysis SE or circularity a perfect circle will have the value 1 and Integrated Intensity Their selection can be toggled on and off EE Eea by a simple mouse click in the selection Set up filters Min Max boxes Selected filters have their check MA 500 1045 S i gt boxes labeled with a tick It is not A E OEE necessary to select any of the filter T Integrated Intensity 58675 parameters if none are selected images Filter will be processed without filtering Reset to Default Values Default values for this imaqe Area 1 4 1045 shape 0 151 0 785 Integrated Intensity 20 0 58675 The text boxes to the right o
461. s This first dialog asks to define the first well edges as shown in this dialog on the left Click on the Record Y offset and X offset buttons at the relevant time 171 Andor Technology Andor iQ USER GUIDE The second skew dialog shown below requires the user to define the top and left edges of the second well The system will move to its estimate of the top left of the second well if you click on the Move to Second Position button to help the user o lt lt Back Next gt gt cet with a starting position When you have identified the top Registration point offsets The skew is calculated using the second well on first row edge click on the Record Y The stage has been moved to its approximate position Offset button and the left edge 1 Steer the stage so thatthe top edge ofthe second click on the Record X Offset well touches the horizontal line button The system will now compute the Skew or 2 Click Record r Offset Y Offset 13 8396 mm Rotational offset for all wells in the pattern and apply this to the 3 Steer the stage so thatthe left edge of the second well coordinates well touches the vertical line 4 Click Record X Offset x Offset 14 7393 mm lt lt Back Nerto Cance The Rejection Inclusion List panel is Rejection List shown next and here you can define Enter pattern for the rejection list which wells should be included in the 2 5 6 Scan pattern This all
462. s 2h Camera Binning Use Current T walt Allocation Off Camera Selection Use Current Events dP Pause Click OK to start protocol ap Repeat T 10 times 2 sec ai Auto Focus Activate Every 2 times Auto Focus ZOC Move Channel Ch al Snap so Move Channel Ch al Snap End TO Repeat T 10 times 100 me vai Auto Focus Activate Every 10 times Auto Focus ZDC Move Channel Ch al Snap a Move Channel Ch al Snap End TU Repeat T 10 times 2 sec asi Auto Focus Activate Every 5 times Auto Focus ZOC Move Channel Ch al Snap Ao Move Channel Ch al Snap i End TO 7 Protocol Test Case 6 Mult Phase with AutoFocus a Image Test Case 6 Mult Phase with Autofocus gt This Protocol contains sequential phases of acquisition with different frame rates in each phase Each phase needs to run with precise inter frame timing but the intervals between phases are long enough for focus drift to be problematic So in this Protocol we introduce Autofocus actions within between phases to maintain focus Note the new Autofocus Activate keyword which allows you to specify the interval between Autofocus actions 212 Andor Technology Andor iQ USER GUIDE 4 6 5 Multi Channel Time Lapse with Z Stacks Z moves first Se wa crotocol Test Case 2 First eg Image Test Case 2 First a Protocol Type Time Series at Camera Binning Use Current 2 Wait Allo
463. s Groups of values are usually separated by tabulations Inside a group commas are usually used to separate each element The beginning of the header contains the name of the image its creation time and some global information on the image format type size in pixels calibration dimensions size Created This section contains the date and time of the creation of the image XYZScan This section contains a summary of the scan pattern ocanName name of the scan pattern FileVersion version number of the header file Centre for internal use only Always false used for backward compatibility only Channel for internal use only Deprecated used for backward compatibility only WellPattern name of the well pattern used only for multi well scans Density density of systematic sampling number of fields per well in systematic sampling mode only for multi well systematic sampling scans 504 Andor Technology Andor iQ USER GUIDE Calibration calibration in um per pixel in use during the acquisition XYFields the number of fields and their position a montage section is not a considered as an XY field See MontageOffsets for montage sections settings A position is composed of X Y and Z Then follows some settings regarding the Z scan for internal use only 6 letters and 1 number Y N deprecated always Y should refer to Z being centered or not P Z B P if Number of planes has been selected Z if Del
464. s existing on the Image Window The Frappa Channels are defined in the Select Frappa Device item form the Wizard menu You can use more than one Frappa device ina system Return to Table Image Edit this item to specify the root name of the image s created by your Protocol Note that this name can be used for AutoSave and so should not use illegal file characters such as V backslash Return to Table Load Protocol Load the named Protocol when the execution of the current Protocol has ended This allows you to switch between different acquisition modes sequentially A different camera can be selected in each Protocol if required It is possible to create endless loops of acquisition which can only be halted by User input or a fault condition if a sequence of Protocol loads the next in a circular manner When a Protocol loads itself this will become an endless loop and can be useful in various ways e g to decimate storage activity with AutoSave enabled or provide bursts activity with known delays or with triggers To make sure that Loaded Protocols do not block execution ensure that no Pause Actions are in the Protocol Trees Return to Table Montage View Creates a new image which is a mosaic or montage of the fields in a Montage scan Can only be instered into the Tree if a Repeat Montage exists in the Tree Its location in the Tree determines the frequency of its execution If within the 196 Andor Technology
465. s that approximately half of the light falling upon it is reflected A and half transmitted The disk is located at an image plane of the microscope optical system so that an image of the SIP is projected into the specimen and about half of the illumination 112 Andor Technology Andor iQ USER GUIDE intensity arrives at the specimen while the remainder is reflected back into the illumination pathway where it is baffled to minimize background In the detection pathway the resulting fluorescence signal comprising what is in focus confocal or C and out of focus wide field or W is imaged back onto the disk where we make use of its transmissive and reflective properties Clara Emission Camera Filters Spinning s Disk Excitation Filters N e lt a DI y 5 Emission ai n Path Reflected NON Excitation Path Emission Path Transmitted The fluorescent light transmitted by the DSD disk comprises the C signal plus about half of the W signal while the light reflected from the disk comprises about half of the W minus the C signal In confocal terminology the SIP is located in a conjugate image plane and hence acts as both the confocal source and detection apertures However as it is not a pinhole we must undertake some further image processing to separate confocal and wide field signals The optical path is illustrated in Figure above green is the excitation path and yellow and orange the fl
466. s the Start position the corresponding End position will be calculated Pressing Select Centre will accept the current position as the Centre position the corresponding Start and End positions will be calculated NB If you adjust the AZ or the Number of Planes then adjustments will be made using the current start position Once the scan positions have been recorded the Move buttons will activate These allow you to move to the Start Centre and End of the Z scan allowing you to quickly verify the Z scan range 4 3 3 Multi Field Scan Multi field Scan definition is the simplest because the system requires no knowledge of stage axes alignment or orientation Multi well and Montage need that information so that iQ can interpret well plate layout and stitch fields together In multi field 165 Andor Technology Andor iQ USER GUIDE scanning you simply record a series of XY positions to be revisited when the Protocol executes File Image List Settings QC Wizards Process Analysis Plug Ins Help Only a summary of the field recording Setup Wizard dialogs will be shown here Choose Setup XYZ options SS Multi field from the Scan Manager and press Next Select Device Device Setup lt lt Back Next gt gt Cancel ee Cancel Enter the desired number of fields in the e options red highlighted text box Select Number of Fields a Press NEXT FIELD to select position and move to next field
467. se from the dropdown menu SOUICE Background Mask Operation Add Diadic Source Operate on two mag Select Runnings UMT Select Select each image by pressing the Select button for both A and B and choosing from the image list 399 Andor Technology Andor iQ USER GUIDE If Operate on two images or image series taking average of secondary is selected DOUCE Background Mask Uperation Subtract Modulus Diadic Source operate on two images or image series A Runnings UM Select B Runnings UM Select Region Of Interest B S i Time For secondary average images Start 177 End 509 Operation Select the Operation from the dropdown list as follows lf Subtract is selected as above picture shows from the on i Modulus dropdown list an additional box will appear giving the user the Signed following options If subtract is not selected Uperation Add no other selections will be necessary 400 Andor Technology Andor iQ USER GUIDE Diadic Source operate on two images or image series Diadic Source operate on two images or image sees A Use the Select button to select an Pensu Salt image from the image list for both A angsuq da an B Region of Interest B When Time is selected use Region Of Interest B dE the dropdown menu to select y Time a Start and End Value For secondary average images Start 177 End 5
468. se until a User clicks on the OK button You can use the message to give instructions to the User or to make sure certain conditions are fulfilled prior to continuing e g adjust some manual equipment such as switching a port or opening a shutter Return to Table Ratio Allows you to specify an on line image ratio When you insert the Action iQ guides you through a setup Wizard in which you define Numerator and Denominator of the Ratio and you can capture two channel images on which to define the Ratio settings With the interactive tools available in the Wizard you can specify the background for subtraction either by stored image or region of interest on the image After background subtraction the ratio is computed pixel by pixel for non zero values and the floating point result is displayed in a second image tab in the Image Window so that it can be viewed during acquisition You can Edit the Action to change the settings if you wish Return to Table Repeat Channel Iterate through a list of Channels held in the parentheses When you insert this Action iQ allows you to choose from the Channel Wizard the first of a list of Channels to be looped This is an efficient way of defining Channel sequencing The right mouse menu on this Action allows you to Add and Remove Channels form the list Return to Table Repeat Time lterate a number of times with a defined interval When you Edit this Action you get a dialog to Edit both number of
469. sedevensdepsansscasudsscusoesnesesaeseassens 495 A3 13 Generic TTL control from Andor iQ cccccscssccsssscccscesccescscccesescccscesccesescccesescccscesccesescccesesoscscesccesess 497 A3 14 Physik Instrumente USB stage controllers ssscccccccssssssssscccccccsssscsscccccccssssscscccsscesssssssscossseees 501 APPENDIX 4 IMAGE METADATA u teieceesetiten cece ceeeti bese eases a aaa a taa aa 503 AA TIQ 1X Image Headers and NCtad ata vccesccccccesscsccisscisccceancacceceescacessaessdetecnvecesiecucdececsuesiviasecceaseevecessscuecess 504 APPENDIX 5 HOW TO DOCUMENTS aaauunaa nua aa aun aa aun aa ait ede eee ede ede esi 529 ASI How to SOC Upa Channel sericis N 530 A5 2 How to Set Up a Multi field Scan sseeecoocsssseeecccssssscccocssssseceoccssssccecsosssssccocosssssceccessssseccossssssscessssssssee 532 AS a HOW 10 Set UPa Series SAAD ATA AT ese e Asara aS raeo aaraa 534 A5 4 How to Set Up a Prototype Protocol sscssssseeecccssssceecccsssssecooccsssseceocosssssccocosssssececossssseeeossssssscessssssssee 535 A5 5 How to Change the Sampling Rate on the Fly sssseecccsssssececccsssscceccosssssececosssssccecossseseceossssssscecessssssee 536 AS 6 Howto Use Load A Erotocol AA ATA ATA E E EA 538 A5 7 How to On Line Chart During Acquisition sscssssseecccsssssecccocssssceeocosssseccocosssssccecossssscecoscssssscesessssssee 539 AS 8 How to Open an Image TIFE Voris aoee a ED DA E EEEE Ee E Aa iaaa NEE aE P
470. settings file to load Pressing the Reset button will reinitialize the filter wheel In the unlikely event that 466 Andor Technology Andor iQ USER GUIDE the Andor Software stops talking to the Sutter Filter Wheel pressing Reset can resolve the problem A3 4 2 Wheel Settings Sutter Wheel A Wheel B Settings The Lambda 10 X controllers can handle up to 3 wheels and two shutters Parameters for controlling the wheels are identical from a user interface point of view and so we describe this once The shutter can be opened or closed manually by selecting deselecting Shutter Open Shutter Open check box The filter wheel has ten positions each represented in the dialogue by a coloured circle If you move the mouse cursor over one of the positions the name and appropriate value e g wavelength of that position is displayed in a popup A left click on one of the positions will move the filter wheel to that position The name and assigned value of the current position is displayed at the centre of the wheel dialogue A right click on one of the positions opens the Filter dialogue in which parameters for that position can be specified The name of the Filter represented by the selected position should be entered into the Name text box 467 Andor Technology Andor iQ USER GUIDE Filter X Name Numeric Value Units TRITC fr Ok Cancel Description F Type an appropri
471. should remain synchronized 2217 Andor Technology Andor iQ USER GUIDE 4 8 Precision Control Unit PCU Triggers and Events see Appendix 4 2 for information on installing and setting up DIO and DAC cards 4 8 1 PCU Box with Digital I O Triggers can be inserted into IQ protocols in two forms Trigger IN and Trigger OUT Trigger IN can be used to start image acquisition or synchronize the protocol via an external source and also to signal an external event and generate an event marker in the image series Trigger OUT can be used to activate external instruments and devices 4 8 2 Andor PCU with Digital I O and DAC Boards In addition to those functions above DAC has the added facility to control additional shutters and a Monochromator for specific wavelength changes during an experiment To simplify connection and protect your system from external catastrophe e g voltage overloads or damage we have designed the PCU illustrated on the next page ves 2 gi PIEZO Preiiision SUN Ut 100 Seer OIEA LACI N DRR i O o The Andor PCU is an opto isolated connectivity device to allow interface between external TTL signals and Andor iQ The PCU box system provides a simple means of delivering TTL inputs and outputs to and from iQ The system includes a PCI Digital 228 Andor Technology Andor iQ USER GUIDE and optionally DAC I O card connecting cable and free standing or
472. sition Auxiliary Devices 6 Now you have installed the ap Parallel port card and assigned its Curent Live snap Record IT Stay on Top Camera Generic Shutter Andor DSD Andor APZ address iQ will be able to write Output Set Up whatever digital patterns you wish ice 1 ar Sl aa Sl Go ge to assign to the TTL outputs of the TriggerName Rede40 device States hich toe X ja ae Open 7 As shown on the left you can edit _ the names and actions of the TT Wh tomticn Openticse O Transition Delay fo ms Interval fo ms 500 Andor Technology Andor iQ USER GUIDE A3 14 Physik Instrumente USB stage controllers Installing the Physik Instrumente driver Use of Physik Instrumente PI USB stages in iQ requires that you install the PIMikroMove software then update the PATH environment variable in Windows to allow iQ to use PI s driver If this is not set up correctly then on attempting to use a PI USB stage controller in iQ you will see the following error Ensure that Physik Instrumente Drivers are installed and PATH set to location of SCSTranslatar Folder Firstly install PIMikroMove from the Physik Instrumente CD supplied with the system If this CD is lost please contact Andor Support During the installation from the CD simply choose Typical setup secondly the directory containing Physik Instrumente s driver files needs to be added to the Windows P
473. sks Q an j luts macros mi Rename this file iy Move this file Do yg gar 1J_Prefs txt j plugins JAR Power rchiver JAR File Text Document D Copy this file 1 380 KB 2 KB A Publish this file to the Web AS SA ImageJ ImageJ cfq Image exe G E mail this file Internet Shortcut CFG File Image Launcher xX Delete this file 1 KB 1 KB Broken Symmetry Software CO README html _ unins000 dat gears Other Places A e a Rene EH B Setup Uninstall 3 DSDProcessing 3 My Documents 4 My Computer My Network Places ImageJ Details File Edit Image Process Analyze Mamme Window Help E Qlalol lt 4 N Ala macros Freehand selections shortcuts Utilities New Compile and Run 3D Analyze Examples Filters Graphics Input Output Macros Registration Scripts Stacks Tools gt v v vv vv vr vv gt i 562 Andor Technology Andor iQ How To Documents 16 The two images opened should show corresponding Penrose dot patterns created at the two locations on the left and right sides of the camera sensor as shown below Choose a bright point near the centre of either image and click the same point on both images Hit OK when complete OK 17 Click on a corresponding dot in the two images as requested as near to the centre of the images as possible then hit OK 18 If successful a Calibration Completed message should be seen 19 Restart iQ and run again
474. solution is maintained in the Y direction The Quad split device produces four images each with half resolution in X and Y The Field splitter optical device splits the image by wavelength and the channel images appear side by side on the camera as shown below 345 Andor Technology Andor iQ USER GUIDE b w a T Automap 100 3 323 5 w e x 500 a The image projected onto the camera and therefore seen during acquisition shows the two channels or wavelengths images side by side as shown above Lateral alignment of the images after capture can be performed by Field Split tool but it is important to follow the manufacturers advice on setting up and aligning the device with the CCD camera first Some suggestions on doing this in iQ are provided in Section 6 3 3 The Field Split tool is accessed from the Process Menu on the Control Window as shown above The Field Split tool is used to separate the side by side channel images into two or four channels by selecting sizing and aligning two or four regions of interest ROI The data underlying these ROIs are used to create a two or four channel image for multi channel viewing and analysis Field Split works on 8 12 16 bit grey and 32 bit color images but not with RGB color images At present Field Split only works on images on the ImageDisk a development is planned to provide Live 346 Andor Technology Andor iQ USER GUIDE 6 3 1 Steps for Se
475. splay Image ff Load Protocol Fura den Store As This first protocol shown left has a Trigger IN inserted at the beginning of the protocol This means the acquisition will be started by a change in signal level as the Axon polarization device activates the trigger when it applies a polarization current to the cell The protocol will run for the designated number of frames until the Trigger OUT at the end of the protocol activates a Picospritzer system to inject a pulse of calcium agonist into the cell 229 Andor Technology Andor iQ USER GUIDE ET Protocol Fura2 ae Following this injection event IQ will A Image Fura Protocol Mi Protea Type ME load the second protocol named ye Events w Pun eo Interval 99 me fastest Repeat 50 times eg Channel Fura380rm 1 api Ea Sean duration monitoring changes of ii A Channel Fura340nm 2 PO Snap Delay 10 me Fura 2 which will run for a set Channel Device Setup intracellular calcium levels within the Wj Dena ese cell At the end of this sequence ani Fura pre drug will be loaded and the Store As protocols pair will run recursively until the user terminates the sequence Insert Item IO Select item to insert To set up triggers in the protocol right mouse click on the place you wish to insert the trigger and from the pop up menu select Insert then Trigger IN or Trigger OUT The Trigger will be inserted
476. splays summary information about the channel as illustrated below Press Close to exit the Channel Manager The channel can now be incorporated into a Protocol Andor iQ USER GUIDE 4 2 2 Important Note EM Gain and Multi Channel Imaging Many Andor and third party systems are supplied with Andor s award winning EMCCD cameras http Wwww andor com When EM Gain is used in multiple channels in the same Protocol EM Gain must be EQUAL in ALL channels EM Gain switching takes time to settling a few hundred milliseconds and if switched between channels may not have time to stabilize before the exposure is started or completed The result will be poor quality or even missing data 159 Andor Technology Andor iQ USER GUIDE 4 3 Scans and the Scan Wizard Acquisition Analysis Mowe Editor Spot Analysis Status ee Auto Say Setup Wizard E c Ando D atash BrowneSAevolution Time Lapse 4utol mages ee SSS Acquisition Calibratiary Sean Manager Optova queo zema 10 897_40xwi Protocol Test Case 1 Frappa Multi Phase Protocol Z Ta Image Test Case 1 Frappa Multi Phase fs RES b Protocol Type Time Series __ Es Protocol SS New Edit Delete Close w Camera Binning Use Current O a Wait Allocation Off Scan Summaries Camera Selection Use Current qa PES Current Scan Blogs Events MFx3F Mon o Pause Click OK to start protocol gt Mowe Channel Ch Scan MFx3F Mon
477. splays the current status of your system A message describes the current status and the box is color coded Pink Idle The system is Idle i e not capturing Cyan Live Live video is currently displayed in the Image Window Yellow Arming system Run has been pressed and system is initializing or waiting for Trigger IN Green Acquiring data Busy State Protocol is running and shows progress Blue Exposing node time remaining If one of the Channels in the Protocol has an exposure time gt 1second Status will turn blue and the time remaining to complete exposure will be displayed The progress of the Experiment is displayed in the progress bar below the Status Box 2 6 1 2 Toolbar The toolbar provides quick access to the Open Open with Imaris Save Live Snap and Channel functions Click on Open or Save to obtain a standard Windows Open Save File dialogue in which the image can be loaded from or saved to disk Click on Open with Imaris to launch Imaris if installed on the system with the current iQ image automatically loaded When Live button is pressed its text changes to Idle and live video is displayed in the Image Window until the Idle button pressed again Live mode is used for interactive focusing of the system and adjusting the imaging device prior to Protocol execution Adjustments to the camera etc can be made directly via Device Setup or Channel Manager 57 Andor Technology
478. stLZ Start Manual Position Set Up Andor iQ and select the Device 325 0 725 0 Setup dialog Unlock the eee io setting controls and select the Wavelength m Voltage c m 81 86 appropriate board In the aa example shown right Bd 1 is Tension Hey ijo Shutter Delay o used as the controller board Controll Board Edk T PCIM DDADEA Version 2 0 Device KI TILL Mono Reconnect 462 Andor Technology Andor iQ USER GUIDE The picture shows Controller Board Selection selecting the board for control of PCU or FastLZ from the Device Setup DAC Board Selection Measurement Computing Corp Panel DEMO BOARD PCIM DDAOB 16 You have finished all the necessary installations The card is ready for use 463 Andor Technology Andor iQ USER GUIDE A3 4 The Sutter Filter Devices A3 4 1 The Settings Page Acquisition Auxiliary Devices xj Sutter Instruments manufacture a BBY eco Nou v range of filter wheels and light sources Sutter Sutter DG4 AOTF Camera Till MIC Prior i for imaging These are supported by Wheel A Settings iQ with two device controllers to allow Status Not Connected l Setting use with the DG4 5 a fast galvo Fiter Wheel IV Wheel A I Shutter A switching source and the Lambda series of filter wheel and shutter Control Port COME MV Reconnect At Start ie devices As with all controllers Speed 4 x Shu
479. sulting in lower intensity values than expected 267 Andor Technology Andor iQ USER GUIDE 5 5 5 Greys per Second When an integrating CCD camera is used to capture data from samples with variations in intensity it is possible to use region analysis to obtain an indication of the amount of fluorochrome present However most systems simply report grey level values without reference to the camera exposure time This is adequate for comparison of samples that can be imaged at the same exposure time but clearly does not take advantage of the dynamic range available when exposure time is varied For example an 8 bit camera has a range of maybe 100 when background level and noise is taken into account but if the exposure time can be varied from 40 ms to 1 second then the range is 100 x 25 i e 2500 In 12 bit cooled CCDs this is even more pronounced Thus if the exposure time is used in the calculation a huge increase in analysis range can be achieved which is a major benefit for scientific applications in areas such as GFP quantification in gene expression For this reason iQ has been given the option of Greys per second f this option is selected then the intensity information is calculated from the grey scale content and the result is normalized divided by the exposure time at which the image was acquired Hence the resultant values are expressed as greys per second and will reflect relative intensities over a greatly enhanced range
480. sv vee Map on mouse up from a sample of the image pixels and 3 sirens adjusts the table to provide a high contrast bright display When checked Automap operates every time the image is updated as in Live or Animation Further options are available when Automap is enabled Use ROI operates on the currently selected ROI for mapping allowing the range to be adjusted as the ROI is roamed over the image Map on mouse up activates an Automap action whenever a Mouse Up event is detected in the Image Window e g when a ROI is moved over a captured image 2 7 1 4 Reduced Flickering Automap now provides additional improvements to reduce flickering when using automap The Smooth option only rescales the image when the min or max value changes by a significant amount configurable in the Options dialog The default change required is 5 The 99 1 contrast option autoscales to the center 98 of pixels This cuts out outlyers in the high and low ends resulting in less flickering and more punchy image 70 Andor Technology Andor iQ USER GUIDE 2 8 The Image Window Menus The Image Window Menus are used to manage images image views and to provide Tools for ROIs used in analysis 2 0 1 The File Menu CFP 16 Bit 672 0 333 x 104 File Edit Image List Region wiew a S Open Image Save Image and Exit Loads and l Open Image Saves images to and from disk and Exits from iQ Sa
481. t All a Focus do Paie tiek O tostat protoca Trees including prototypes have a series ae of Actions which cannot be removed from ag Auto Fog Edit ery 2 times Jo Flepeat Insert the Tree They are used for system level RA Auto Delete 1 l l Repeat Montage control settings and some can be edited Montage Positions 4 2 by 2 J 27 Positions 3 Repeat 9umin 17 planes at Move Channel GFP as discussed in section 4 4 4 gl Snap Selecting Edit on an Action item iQ2 will End Z l End Montage show an Insert Item dialog section 4 4 3 End sY End To from which you can choose or a dialog allowing text or value entry To Insert an Action keyword into the Tree you point to the Action above the insertion point and right click the mouse A popup menu will appear as shown to the left and the new Action will be inserted directly below the selected item in this case at the Repeat Time Action 191 Andor Technology Andor iQ USER GUIDE In general insertion rules have been minimized to support Protocol flexibility Where rules do exist they are expressed by restricting the list of Action items presented in the Insert Item dialog 4 4 3 The Insert ltem Dialog The Insert Item dialog shows the list of Actions which can be inserted at the selected point in the Tree Each Action keyword has a name an associated icon and a text Hint to help you see its function at a Insert I
482. t Dual Camera File Folder ES Common Test Mem Leak File Folder O Configuration Test New Config File Folder ie DLastSession ini 4KB Configuration Settings E O iex E LastSession rgn 25KB RGN File D Dummy G Fiz I ixon id 4 b ii 4 2 1 objects selected g My Computer A Select sort by date find the latest files and look for the file MyTemplate Kl_EXP and copy to the clipboard You may need to activate the file extension in windows explorer see Windows help for details t iXon Prior X81 Piezo ALC Frappa File Edit View Favorites Tools Help Q sax 7 O f 5 Jo search gt Folders ES rs x 1 Ea Address L G Documents and Settings All Users Application Data Kinetic Imaging iQx ixon Prior IX81 Piezo ALC Frappa Folders E MyTemplate KI_RATIO 1KB KILRATIOFile 12 13 2010 9 46 AM E ES iex Dummy E MyTemplate KI_NODE 3KB KI NODE File 12 13 2010 9 46 AM FLZ MyTemplate KI_MERGE 1 KB KI_MERGE File 12 13 2010 9 46 AM EI ixon Ed MyTemplate KI_LZEXP 7 KB KI_LZEXP File 12 13 2010 9 46 AM ixon ALC Trigger MyTemplate KI_EXP 8KB KI_EXP File 12 13 2010 9 46 AM ixon Ix81 ALC Piezo FRAPPA Prior MyTemplate ChartOptions 1KB CHARTOPTI 12 13 2010 9 46 AM o iXon IX81 ALC Piezo Prior Frappa LastSession KI_RATIO 1KB KI RATIO File 12 13 2010 9 51 AM 3 ixon Micropoint LastSession KI_NODE 3KB KI_NODE File 12 13 2010 9 51 AM oe iXon Prior 1X81 Piezo ALC
483. t Use separate image if you would like to create a mask with another image Use the select button to select another image from the image list select the Create Mask Image to create a mask This will launch the Mask Wizard Section 6 1 2 404 Andor Technology Andor iQ USER GUIDE 6 12 4 Processing Summary Each option selected under the Source Background and Mask Tab will be previewed at the bottom of the Math Plug In Window as shown below Source Background hlask Background A Ho Mask Background E Under Source there will be a preview picture for each image chosen However if Operation with N dimensional TIFF or Self Operation taking average of selected range for secondary is selected there will only be one image present under Source Background will preview the selections chosen under the Background tab for both Background A and Background B The selected Operation e g Addition will show in the circle The Threshold values for the Mask for both A and B will show under Math if a mask is selected Close Press the close button to exit the Plug In Help Press the Help button to access section reference for User Guide Process Once all selections have been made under each tab press the Process button A new window will open as shown on the right If Display Current Image Slice is selected a live preview will show in the window Press Cancel at any time to exit setup The new image will then
484. t Z Image Math Rato Image Image Image Math Dual Image Disk iQ s Main Window It could be used to Isolate single molecules from the background in fluorescence or to correct for uneven illumination in brightfield for example 1 In the above illustration the concept of a Rolling Ball is shown The black line represents the signal intensity in the original image A ball is rolled along the signal and the centre of the ball forms the signal in the new image Any signals smaller spatially than the diameter of the ball are rejected irrespective of their intensity This can be useful to isolate small objects from background reject small objects or to smooth intensity changes across an image Rolling Ball Filter A fe Dark features fe Result Radius 50 C Bright feature Background Subtraction v F ikani Original Image Processed Image Andor Technology 360 Andor iQ USER GUIDE When the Rolling Ball Filter plugin is started the window will appear and the currently loaded image will be displayed in both the Original and Processed section Note that the Processed Image section is for preview purposes only and may be subject to mapping or contrast changes In this image it can be seen that the background of the gel is unevenly illuminated To correct for this a radius larger than the details that should to be kept should be entered in the Radius textbox Because this is a brightfield ima
485. t possible before We hope also that the GUI improvements make iQ2 easier to use and more intuitive and make the time you invest worthwhile Our discussions with beta users and our internal testing schedule Suggest that we are on the right path with iQ2 We welcome your feedback comments and suggestions 218 Andor Technology Andor iQ USER GUIDE 4 7 Multi camera acquisition In versions of iQ since 1 8 we have provided support for handling multiple cameras on a single Windows workstation In the 1 10 2 release we improved performance and management of multi camera scenarios as this becomes increasingly popular A single instance of iQ allows the control from two or more Andor cameras but not third party products IQ can be configured to allow sequential operation of cameras in a single instance or parallel operation of cameras from multiple instances one camera per instance This document presents a summary of how to set up systems for multi camera applications in the following scenarios e Dual camera sequential use e Dual camera simultaneous use e Adding a third camera in these cases Recent developments in multi core processors and fast hard drives means that a single high performance works station can provide the necessary bandwidth for simultaneous multi camera acquisition where in the past multiple workstations were required Current generation iQ workstations have the capabilities needed to support multi camera acquisition
486. t section below it becomes available allowing the choice of whole image normalization or normalization using the currently selected ROI in the Image Window 343 Andor Technology Andor iQ USER GUIDE 6 2 Mask Wizard Mask wizara lll Mask wizara lll Setup Wizard In image analysis it is common practice to Es isolate features of interest for subsequent measurement This tool enables a binary ema Please select the Image Source mask to be created which can be applied to O Ne xt gt gt any image using standard mathematical WINETSERVERSDEMOIMAGESCA RATIONFURA TIF14 Select Name FURA TIF14 Type 12 bit grey pseudo A tensity 1 000000 lux 416 1 000000 pm 7 length 2 nn aveleng 2 nm Time 200 ms operators prior to image quantification The Mask Wizard is accessed from the Process menu on the control window Clicking on the selected item launches the set up wizard shown below Following the execution of a pre processing algorithm a user defined threshold is applied to the source image to create a binary mask The functionality of this module is identical to that discussed in the section describing the use of the Ratio Tool Further details concerning the use of the Mask Tool can be found in Section 6 11 344 Andor Technology Andor iQ USER GUIDE 6 3 Field Split The Field Split tool is intended for use with optical devices Episod
487. t_ F Multi CCH aia neo Resin Gx pa Set Defaut 100 148 23 0 i e o al 5 7 6 6 Clear Data Clicking on the Clear Data button removes analysis data from memory following confirmation by the user 5 7 6 7 Save Data The Save Data button is used to save feature data to a results data file 304 Andor Technology Andor iQ USER GUIDE 5 7 7 Multi frame or Time Series Data Additional functionality is available for the statistical analysis of time series data Pressing the Show Data button displays the following data table File Plots Data View Frame Area Perimeter Max Chord Shape Int Int Mean Int Loglntint 4 m Cei Jo 552 000 110 000 31 000 Cel2 1 868 000 152 000 49 000 0 472 119806 000 138 025 11 694 Cel3 1 591 000 118 000 36 000 0 533 94156 000 159 316 11 453 Cel4 1 557 000 116 000 35 000 0 520 89870 000 161 346 11 406 Cell 5 2 570 000 116 000 35 000 0 532 63679 000 146 805 11 335 Cell 6 2 539 000 116 000 36 000 0 503 79624 000 147 725 11 285 Cell 7 2 689 000 144 000 41 000 0 418 102138 000 148 241 11 534 Cels 3 506 000 116 000 32 000 0 473 68472 000 135 320 11 134 Cel9 4 821 000 136 000 41 000 0 558 98114 000 119 505 11 494 Cell10 4 893 000 134 000 38 000 0 625 110248 000 123 458 11 610 Celli 4 513 000 110 000 34 000 0 533 42553 000 62 949 10 659 Cell12 4 564 000 120 000 35 000 0 492 5622 000 98 621 10 926 Cell13 4 551 000 110 000 31 000 0 572 56900 000 103
488. ta Z has been selected B if End position has been selected STEPO kept for legacy has no meaning anymore For Z single field scans no information is displayed here as we don t store the X Y position of the scan ex 3 8594 656 12117 763 171 65 YPSTEPO 12211 789 12260 757 175 75 YPSTEPO 9979 865 14038 272 168 85 YPSTEPO0O ZScan STEP followed by the size of a step then the number of steps In Z single field scans this is preceded by the center of the z range followed by settings regarding the Z scan see XYFields ex 189 99 YZSTEPO STEP50 4 Montage the type of montage Region Edge Linear Overlap followed by the overlap between montage sections in percent Width followed by the width of a montage in um Height followed by the height of a montage in um Units followed by the unit used to define the montage in iQ Fields or MM ex Region Overlap5 Width 582 32 Height 581 16 Units Fields Plates the number of plates and a description of each plate Each plate contains the following information X and Y of the top left edge of the top left well X and Y of the top left edge of the second well of the first row rejection list inside square brackets the first well s ID is 1 wells are separated by comma a boolean for the rejection list 1 if itis a rejection list O if it is an inclusion list MontageOffsets the number tiles in a montage and the respective offsets of each tile i
489. te option from the Disk Selection region will update the list Any image can be copied from the Image Disk shown on the right to the Local Disk by pressing the lt lt Copy button although this step is not necessary to combine images Combining two images together is simply a matter of selecting one image from each Image Disk and pressing the Construct button This will copy the selected image from the Remote Image Disk to the Local Disk and combine them in one operation The name of the new image can be defined in the Name textbox The new image will appear in the Local Disk and can be loaded into the Image Window by selecting it and pressing Show Note that images to be combined must be identical in all dimensions e g it is not possible to combine one image that is 1000 x 1000 pixels with one that is 512 x 512 pixels nor is it possible to combine one image with 100 time points with another that has 101 time points even if the resolution is the same in both images Finally it is possible to perform an alignment during the construction process To create the alignment file first run the Field Split plugin see Section 6 3 and save an alignment file To enable alignment during construction check the Apply checkbox in the Alignment File section of the Dual Image Disk window and open the appropriate file by pressing the button to the right 408 Andor Technology Andor iQ USER
490. ted step 3 carefully and systematically lock the barrel set screws checking that alignment is maintained Minor adjustments to the cassette system may be needed once external supports are tight 5 Use matched intensity images for software alignment Snap images of closely matched intensity on each system for the software alignment step if you have to adjust exposure time that is OK for setup Use the Dual Image Disk Plug In on the Master system to connect to the Slave system and show the two Image Lists See chapter 2 for information on setting up a Dual ImageDisk configuration for use with two cameras and two PCs 6 Make sure both lists have been Refreshed select the two images at the top of the Image Lists These two images will be merged into a dual channel image on the Master system which will be used to create the software alignment file 121 Andor Technology Andor iQ USER GUIDE This dialog shows the Dual ImageDisk dialog which is accessed from the The red ellipses high Process Menu light the Refresh buttons which update the lists The Constructor button frm_DuallmageDisk Local Disk RG dual_split RG dual RFP55 GFP53 dual test_split dual test GFP522 dual rot_split1 dual rot_split dual tot GFP501 GFP50 GFP49 Name Construct Duana ment File Testdligment51G52 4LI 1 Dynamic Image Disk C Remote Image Disk 2 highlighted with green
491. tem glance When you click an Action item in Select tem to insert ENET the list a text Hint is shown at the bottom isl Lio FOC WE mi Auto Focus Activate of the dialog as highlighted left We hope P Channel Repeat chy Delay the Hints are instructive and clear but If Frappa cee as ia Move Channel you need more clarification please read on Move Channel ANO Snap FP Move XY where we provide more details for each gt Move zZ Pause 2 Snap Action EN Time Repeat Trigger In IMPORTANT NOTE When you insert any Trigger Out i l amp Z Repeat Repeat Action an associated End Action will be included immediately afterwards You can drag this End Action downwards to enclose the Actions you want to enclose Hint Iterate through list of Channel e g Channels Snaps etc Help Cancel A List of Action keywords and their Hints is shown in the next section and from there we explore each Action in more detail 4 4 4 Action keywords an overview ltems here are coded as follows Red Always in the Tree but not in the Insert Item list Blue Added to the Tree as part of an XY Scan User can Edit via a popup menu on any XY Scan component blue Find the Action keyword in column 1 and then click on column 4 with CTRL key for 192 Andor Technology Andor iQ USER GUIDE
492. the event after which you can stop the experiment lf Display Image is selected live video is displayed in the Image Window during the image acquisition If de selected live video is not displayed This option has the advantage that the image acquisition is quicker The Store Store As buttons are used to save Protocols The information in the Protocol Command Tree will be loaded saved 60 Andor Technology Andor iQ USER GUIDE 2 6 1 6 Protocol Tree You create Protocol Trees from the Protocol Wizard using New But you configure the details of Tree Actions with the right mouse menu which shows Edit Insert and Delete when the context allows it Tree Actions are placed inot an execution stack when iQ prepares to Run the Stack allows iQ 2 to build complex Protocol construction protocols with Repeat loop nesting to many levels subject to final image dimensionality not ten If a Tree Node has a plus sign to its left a mouse click on the will expand the tree as shown here A right mouse click on an Action in the Protocol will open a pop the that command are displayed For up menu in which appropriate options for example right clicking on a Channel node will display the following options fa 2s Focus dP Pause Click OF to start protocol Repeat T 50 times 1 min aai Auto Focus Activate Every 2 times o Repeat sir A Auto Focus Motor Repeat Montage Monta
493. the drop down list box in this case a 8 position XY scan was chosen After you click on Next the Scan will be inserted into the Protocol Tree as shown on the left Note when you Insert the scan into the Tree iQ will either update or initialize the scan graphic with the well pattern if applicable and the fields to be scanned as shown here lt gt Included Vell Region Current Position Rejected Vell Region 184 Andor Technology Andor iQ USER GUIDE 4 3 Adjusting Scan coordinates using the Protocol Tree Scan coordinates can be adjusted easily using the Protocol Tree It is not necessary to resort to the Scan Wizard to modify an existing scan pattern scan coordinates can be modified by a click of the mouse 4 3 7 1 Modifying a Multi Well Scan The Protocol Tree corresponding to a Multi well scan experiment is illustrated below a thy Protocol protocol A Image image ame fF Protocol Type Scan Protocol N Run Channel The details of the scan pattern are disclosed by clicking on the Lou uj Pause Click OF to start protocol SF Interval 1056 ms fastest epee Repeat 7 times Bes Repeat Macro Bi Scan Pattern 6 well costar i ET well 3 PME ee YS 27625 11341 0 ie JP 8 6 38574 11341 0 oon D Y 7 49522 113410 ke O XY B 27625 5040 0 ET Well 3 EI weld EI Well 5 ET well6 AACS Repeat Wr lf End Ma
494. the DSD calibration alignment process and this time you should have success 20 If either the Land marking or subsequent alignment calibration in iQ does not succeed then you need to contact Andor support for further assistance 563 Andor Technology Andor iQ How To Documents A5 19 How to Setup MicroPoint for Uncaging MicroPoint is an extremely flexible tool for photo stimulation delivering precisely positioned laser pulses over a wide wavelength and energy range Depending on energy and wavelength setting the device can be used in order of decreasing energy to perform the following e ablate cells and organelles e bleach fluorophores activate photo switchable fluorophores and release caged compounds breaking molecular bonds The Micropoint 1 system is driven by a pulsed nitrogen dye laser This laser has some patented features that allow it to deliver about 25 different wavelengths determined by the contents of a dye cell resonator The dye cell contains various mixtures of mainly coumarin dyes which fluoresce strongly and in combination with the resonator provide a tunable pulse delivery system The nitrogen laser provides driving energy in 70 uJ 3ns pulses at 337 nm and at 10Hz These pulses are converted in the various dye cells to wavelengths in the range 365 650 nm with duration of about 4ns The energy delivered to the specimen can be varied from about 50 uJ down to pu levels When using the system at visibl
495. the features displayed in the Image Window Pressing the Get Navigator button records the position currently shown in the Image Navigator This function can be used to adjust the position of the Min and Max slider bars Similarly pressing the Set Navigator buttons updates the Image Navigator with the information represented by the position of the Min and Max slider bars The new sub sampled image is copied to the Image List by pressing the Create button Clicking on the OK button exits the Selector tool ignoring any changes to the Selector dialogue panel 2 8 3 The Image List Menu This opens the Image Manager which displays all images in memory manager together with image information This has the same functionality as in the iQ Menus Section 2 6 2 79 Andor Technology Andor iQ USER GUIDE 2 8 4 The Region Menu Paula drosophila PBC_VerticalFlip_Rotate180 16 Bit 642 0 107 x 1070 0 107 4D File Edit Image List Senin View Fast MIP ok qe ce Time Save Spot Region Colour Options The Region menu item enables ROI s on the image to be assigned to a particular Class and to be saved to disk from where they can be subsequently reloaded Classification this assigns the selected ROI to a particular class the current ROI class is displayed in the Image Toolbar as illustrated below The ROI you wish to classify is selected by clicking on it using the Hand tool Section 2 10 1 or the Pointer to
496. the focus because of thermal drift The acquisition can be interrupted by pressing the Live button and refocusing the image The effect of refocusing is to alter the coordinates of the scan The acquisition is restarted by clicking on the Idle button after which the scan is reset to the first field and the Z Stage coordinates at all XY positions are modified by the offset introduced in the refocusing step A revised image series at that time point is acquired Please note the resultant image data does not contain the new position stamps corresponding to the revised positions in the Z series The Z positions calculated at the start of the acquisition are used For multi field or multi well protocols the right mouse action on the current field is used to update the X Y and Z values This can be done sequentially on each field of the scan to ensure good focus throughout 188 Andor Technology Andor iQ USER GUIDE 4 3 9 Editing a Systematic Scan The coordinates for each field in a systematic scan can be recomputed based on uniform random sampling using the Protocol Tree The scan pattern is highlighted using the left mouse button and the Edit function accessed by clicking the right mouse button Clicking on the Edit function launches the Scan Manager setup wizard where modifications to the acquisition channel scan pattern type and number of sampling fields can be made lf no changes are made to the scan pattern and the current num
497. the histogram If you wish to set the current values as the default values click on the Set Defaults button To reload the default values at a later date click on the Get Defaults button To save the histogram data i e the number of cell nuclei in each histogram bin and the displayed histogram statistics press the Save Histogram Data buiton A standard Windows Save File dialogue box is opened which you can use to save the data in the usual manner The data file can be read into standard spreadsheet programs such as Microsoft Excel 297 Andor Technology Andor iQ USER GUIDE Save Histogram Data Save ir A Kinetic a colona slh Save as type Spots Excel histogram data slh Cancel The Print Graph button enables the charted data to be printed for further inspection and comparison An example Print Histogram window is illustrated below Bie ES Close Print Histogram Printer WEB SERVER Brother HL 1 Setup Print Orientatior i Portrait i Landscape Reset Margit M View Margins W Proportional o AR ee The Save Histogram Data and Print Graph functions are also accessible from the menu bar File Plot Save Histogram Data Print Graph pie The Close button exits from the Histogram dialogue and returns to the Data Review 298 Andor Technology Andor iQ USER GUIDE panel This function is also accessible from the menu bar Histogram of
498. the joystick move the XY stage to lt lt Back Newt gt gt c the position of the first field in the first well Multi_Field options The X and Y positions will update in the text boxes as will the Z position if you have a Z Press NEXT FIELD to select position and move to next field moto r Fress PREVIOUS FIELD to select position and mowe ta AOUE TEN Press Next Field and then repeat the Please ensure image is focussed as z position will dig be recorded f available above to specify the positions of each field x position Current Well 1 for all wells SAME AISI At the start of each well the stage will y position lo j 9 lt lt Previous Field NewtField gt gt automatically move to the top left quadrant iti fo n of that well Multi well Montage aloj x When you enter the field EELS 1g a ecm cate accent oma cana definition phase the scan graphic will show the well 150005 RUA AA A ey ea eae pattern and the fields within the e ATE PISO ira Sa SA A wells as shown on the right 18 000 gt This example shows a 24 well none i gey plate with systematic i sald ee a ee sampling density 5 fields per annie a wand trary eee Gant well Note that wells 9 10 15 m and 16 have been Rejected 12 000 14 000 16 000 18 000 20000 22 000 24000 26 000 The First well is shown at top left and identified as blue while remaining wells are green Note Autofocus when you want to record and
499. then choose Setup More details of the Ratio action are described in Section 4 8 2 4 For the most precise processing of Fura and other ratio image data iQ provides the Ratio plugin A detailed example of the use of this plugin is presented in Section 5 8 204 Andor Technology Andor iQ USER GUIDE 4 5 2 1 On line Charting The On line chart tool enables acquired data to be visualized in real time intensity parameters are updated live during the course of an experiment Data charting is enabled by inserting the On line chart keyword into the Protocol Tree Chart Options Channel Ratio Chart Options File Streaming A Axis E Background Correction During Acquisitiorr During Animation Ok Group Regions AE C Time C Dimension Separate Plots ve y Cancel Show Legend fe Frame Number Frame Number Show Event Labels Load Region Display Chart History All Regions Enabled So ES Selected Region Scaling Channel Ratio fe Auto Scale fe Auto Scale C Manual Min 0 Manual Min Max 256 Max 3 When the On line chart is in Setup mode the user is shown the dialog above to specify the channel or channels to be charted under the Channel Tab A Ratio chart can be selected using the Ratio Tab Expansion of the Protocol Tree in the example illustrated shows the inserted Online Chart When the protocol is executed the on line chart is displayed on the screen and the time interval over which th
500. tical data for one user defined region of interest The following data is calculated 250 Andor Technology Andor iQ USER GUIDE Mean the mean intensity of the grey pixels Peak the peak intensity of the grey pixels SD the standard deviation of the grey pixel intensity Area the number of grey pixels within the region within the threshold limits specified in the Settings Dialogue Area the percentage of the area within the threshold limits specified in the Settings Dialogue Integrated Intensity the integrated intensity within the threshold limits specified in the Settings dialogue Integrated the percentage of the integration within the threshold limits specified in the Settings dialogue Minimum Threshold the lower pixel intensity threshold limit as specified in the Settings dialogue Pixels with an intensity outside the minimum and maximum threshold values are ignored Maximum Threshold the highest pixel intensity threshold limit as specified in the Settings dialogue Pixels with intensities outside the minimum and maximum threshold values are ignored Region defining coordinates the X and Y coordinates of the top left bottom and right points of the region 253 Andor Technology Andor iQ USER GUIDE 5 4 2 Channel Statistics The Channel Statistics Tab opens the Channel Statistics page Statistics can be calculated here on the user defined regions for each of the filtered plan
501. ting methods It provides an optional normalization correction method either from the pre bleach or from a separate reference region Results are calculated either from the original or corrected data fits depending on what correction mode the user has chosen The user can select combinations of points in the multidimensional image acquisition and define any number of FRAP periods within each region 5 13 1 Terminology lt is useful before proceeding to explain the following terminology Point Position A point in the image dimensions from which a time series can be plotted Region Used in two contexts o An ROI set on the image view o Acurve displayed in the analysis dialogue There are two types of regions o FRAP Region Within which the FRAP occurs o Reference Region An optional region which provides data for correction FRAP Period A FRAP Region can contain any number of FRAP Periods The duration of an entire FRAP event Andor Technology Andor iQ USER GUIDE FRAP Interval A FRAP Period always consists of four FRAP Intervals The time period over which a characteristic FRAP sub event occurs There are four different intervals o Pre bleach o Bleaching o Recovery O Ste ady State Real Time ms 5 13 2 The FRAP Analysis Dialogue The FRAP Analysis dialogue is opened from the Analysis menu in the main iQ window It is dynamically populated depending on the user experiment An example is shown below An
502. to use including the ALC with MPU selected camera and CSU B Make sure that you have a suitable sample for photo bleaching either Andor Test Slide 001 or a fixed Molecular Probes specimen e g Cells 2 where microtubules are labeled green excited at 488 nm Alternatively Mosaic can use the fist surface mirror that is provided with the system in order to properly calibrate the system Mosaic illuminates a series of points or patterns that are reflected off the mirror and used for the calibration routine C Start Andor iQ 2 or later exe dated July 28 10 or later D Make sure that Mosaic is connected on a suitable COM Port Note the FRAPPA calibration is independent of objective magnification but depends critically on the camera orientation alignment and pixel organization e You may wish to create a Frap channel for Mosaic as with the FRAPPA but this is not essential as the Mosaic device is not in the light path and you may use the current channel settings to Calibrate 2 Run the FRAPPA Calibration Wizard from the Wizards menu If there are multiple FRAPPA devices in the configuration select the Mosaic FRAPPA radio button to calibrate the Mosaic device Choose to create a New FRAPPA calibration or choose the FRAPPA calibration you want to Edit 3 Next you may select the Manual checkbox if you wish to manually select a range of positions points to calibrate but we recommend you keep the Manual checkbox de se
503. tom installation option i Sentinel System Driver InstallShield Wizard Setup Type Choose the setup type that best suits your needs Please select a setup type Complete All program Features will be installed Requires the most disk space Choose which program Features vou want installed and where they Will be installed Recommended for advanced users Installshield lt Back Cancel Andor Technology 419 Andor iQ USER GUIDE Disable the USB Support Not available for users in NT i Sentinel System Driver InstallShield Wizard Custom Setup Select the program Features you want installed Click on an icon in the list below to change how a Feature is installed ee M ERRE be USB System Driver This feature will be installed on local hard drive Install this driver For use with the raal Description LSR SunerPra hardware kew ESE This feature and all subfeatures will be installed on local hard drive 4 This Feature will be installed when required This Feature will mot be available Install ta C Program Files Rainbow Technologies Sentinel System Driver Change Installshield Help Space lt Back Cancel Continue with the installation jie Sentinel System Driver InstallShield Wizard Ready to Install the Program The wizard is ready to begin installation Click Install to begin the instal
504. tput Set Up the LLE is in External control 0 1 3 4 5 E mode Toggle the TTL output by clicking the buttons shown on the States left side of the dialog below Clicking on Low will set D2 ele High Cyan to On while clicking High will set it High and switch it off AU High Low set an Interval to e g 1000 ms UU Low High and when you click on Low High Transition Delay the Cyan source should pulse on Interval for 1 second 575 Andor Technology Trigger Name Cyan 480 fan O fa Pp qu foo x 576 Andor iQ How To Documents 7 Andor iQ 2 2 Kinetic Dummy Camera Fie Image List Settings Process Analysis g Help If all is working move on otherwise Seiwa check hardware connections and state Setup FW and Shutters in Device Manager If there is a for GFP problem highlighted with an explanation mark then check with the ceBeck Newt gt gt hardware manufacturer website for driver updates or Known issues Pre Frame Action Current Shutter 1 Current Shutter 2 Current Current Shutter 4 Current Shutter 5 Current Shutter 6 Current Shutter Current With the control working you now setup Channels for imaging Setup Channels through the Channel Manager as usual and you will see the Post Frame Action Current Shutter 1 Current Current Current Shutter 4 Current Shutter 5 Current Shutter 6 Current Shutter Curre
505. trip can be recorded as a full dynamic range image in iQ by clicking on 324 Be time 0 00 00 028 ae a time 0 00 00 029 9 time 0 00 00 032 Using the interactive dialogs above and left you can choose the dimension in which you wish to iterate in this case Time and the start and end frames and frame interval to be rendered You can choose the size of each frame and the number of columns in the rendered frame matrix You can choose to show the dimension stamp this sequence shows frames 26 33 in a 4x2 image matrix and the frame number is shown as an overlay on each image frame The background color orange in this case can be user defined click on the Colour button as well as Border width separating each frame Andor Technology Andor iQ USER GUIDE the Snap button at the top right of the Strip View dialog For best presentation quality keeping the border color and dimension stamp information you should use the Alt PrintScreen key combination This will place the bitmap image into Windows Clipboard so that it can be pasted inot your presentation direct or into a graphics application e g Windows Paint for saving as JPG or similar format Strip View CLY 647 Animation Sele Update Image Depth Dim Time 0 x j Draw Slices Snap SU nod time 0 00 22 364 time 0 00 22 365 time 0 00 22 366 time 0 00 22 367 time 0 00 22 368 time 0 00 22 369 time 0 00 22 370 time 0 00 22 371 Strip
506. tter Delay fo ms Close Shutter On Move accessed is via the Device Setup dialog The figure left shows a Reset Create Log File Log File c Sutter log Load Save Command Status Ibl_Diagnosis e a Save Buttons allow you to maintain details of filter information loaded into the wheel configuration with both a Sutter tab and a Sutter DG4 tab illustrating that these can be controlled together The Settings page is used to Select the device type speed shutter delay and COM port for control Load and Settings Filter Wheel Lambda 10 7 Lambda 10 From the Filter Wheel dropdown Lambda 10 2 Lambda 10 3 Lambda DG 4 5 listbox select the filter wheel controller Control Port that you are using Speed 4 x Shutter Delay fo ms Close Shutter On Move Available options are Lambda 10 controls one filter wheel only 464 Andor Technology Andor iQ USER GUIDE Lambda 10 2 controls up to two filter wheels Wheel A and Wheel B and one Uniblitz shutter Lambda 10 3 controls up to three filter wheels Wheels A B C It can also control two shutter on ports A and B DG4 5 the fast galvo based light source with four or five positions Switching time is lt 1 2 ms and shutter time is 500 us This device can be driven from a separate controller with the same functionality You will choose that controller if you also want to run a filter wheel with this controller
507. tting up the Field Split Process Open a suitable image from the ImageList Once image is loaded select Field Split form the Process menu options Note If your source image is a Z stack you should select a well focused Z point in the Image window before starting the Field Split Process as this will make the alignment process easier The Field Split Window will open its own window and display with two rectangular ROIS in the window one in blue color labeled r1 the other yellow labeled r2 as shown below Field Split Dual tif l0 x Alignment y EEN lt be ROTO Rol ae v Load OK Close 4 The Field Split Tool Shows o a r2 the two channel ROls in Yellow and Blue and the Line profile option overlays the right and left channel data to allow alignment on a feature which is visible in both channels AR en During this process the Width 240 x B x2 252 7 Profile x xp 55 a a a m o Height 490 vt Poo y2 fr F Profley Yo 154 Red id a original Image In the Image Window has been replaced by a new two or four channel image which is formed by the two ROls as two channels as shown in below Note You will need to use a calibrated multi fluor bead specimen or your judgement to confirm if the new dual channel image is adequately aligned You 347 Andor Technology Andor iQ USER GUIDE should observe the change on the Image Window while adj
508. ttons A typical 500 1 000 1 500 2 000 2 500 3 000 histogram from an Area Time resolved data di i d ti Statistics Histogram Data Global Main Peak C All Dat individua ime Gilets Maines O mmn AID aa all 504 Max 3781 Animate Delay 0 5s frame IS shown Max 3781 ii Bins 20 Frame 18 Mean 1051 55 Close C Statistical plot 677 102 Update Display SD 411 I Mean I SE below Re Tein v Iean LO Cy 64 4 62 8 cies 5 vex Twin io longer Print graph Spots Set Defaults e SACAR Ti After changing Min Max or the number of bins in the histogram click on the Update Display button to update the histogram If you wish to set the current values as the default values click on the Set Defaults button To reload the default values ata later date click on the Get Defaults button The Print Graph button enables the displayed histogram to be printed for further inspection and comparison An example Print Histogram window was shown 308 Andor Technology Andor iQ USER GUIDE previously in this section When the Save Histogram Data button is pressed the number of features in each histogram bin for each time frame in the temporal data series is written to a data file together with a summary of the histogram statistics save and Print functionality is also available from the menu bar as described previously 5 7 7 2 Time Series Plots Clic
509. u If there are multiple FRAPPA devices in the configuration select the MicroPoint FRAPPA radio button to calibrate the MicroPoint device Choose to create a New FRAPPA calibration or choose the FRAPPA calibration you want to Edit 3 Next you may select the Manual checkbox if you wish to manually select a range 141 Andor Technology Andor iQ USER GUIDE of positions points to calibrate but we recommend you keep the Manual checkbox de selected as this will automatically provide a list of positions points which will deliver the best calibration for the device range e FRAPPA Calibration BHA 4 The Calibration panel allows you Help Setup Wierd to calibrate how the device range FRAPPA Calibration o translates to camera pixels This Setup FRAPPA alignment and calibration settings ensures that interactive FRAPPA operations can be executed at the correct position Below the combo Next gt gt Eancel box of pre defined device positions if Alignment Steps Auto Increment is checked the To align the FRAPPA unit use a device will automatically move to the specimen which can be bleached e g Andor Slide 1 next position when the Add Calibration Point button is clicked for Step 1 Annotate Add descriptive comments the current position If Bleach on Bis jie ie fies pl il een Selection is also checked the new Step 3 Bleach Focus on a uniform fluorescent region position will be bleach
510. u will see the channels overlaid and any shifts between the two will be apparent In the main iQ Image Window two rectangular regions will appear on the image and these can be moved around to adjust the relative shift between images The 4 way arrow buttons are used to shift them in one pixel increments or the regions can be Ts g n re eon mm raios e E Green 7 Profile Y Yp 246 dragged on the Image Window if large steps are needed Field Split Dual P d Alignment Load Save 9 Check with Difference image you want to make sure the alignment is optimum use the Diff check box at the top of the Plug In This creates a difference image in the display and also computes the maximum difference in this image You should try to minimize the Difference image to obtain the best m atch Width 423 raw e A x 4 Wavelength e00 nm I Profile XP 246 Height 428 Flip Rotate 4 23 v 2 Green x ax Di I Profley Yp 246 Save in Alignment gt e amp KE z 3 dual 1 ALI 10 Save the alignment file Once you have the roaa w Fliprot 2ch ALI LastSession ali best result you can achieve use the Save aitestalomeressiese at button at the top of the dialogue to choose a Filename E4ampe III name and Save the alignment file You will use O PO
511. uffice because this is unique to the z dimension The units resolution and origin may also be specified although this is not necessary to import the image Click Finish 543 Andor Technology consists of only a Z dimension Click Next Image Importer Wizard Image Import Options Set Time and Z lt lt Back Finish Cancel You need to input Time or Z dimension Identifier in this page The identifier should be the text string that precedes the numerical index for the dimension Setz Z Identifier fz Unit Resolution Origin Files S MULATED_PSF_z0000 tif F Andor iQ How To Documents A5 11 How to Adjust the File Size Limit 1 Select Settings from the File menu in the iQ Andor 10 Control Window Blew Image List Settings OC wWizar Open Image Save Image Set Save Path 2 The following dialogue box will appear pile Settings Orca ER L Si tedimage 3 3 4 g xl set Single File Size Limit ES l Type 16 bit Single File Size Limit 1 2000 MB J iat eee og Image Size 256 x 25 2000 UME OF Cancel 3 Enter a new maximum file size and click OK Click Cancel to close the window without making any changes The maximum single file size supported by iQ under Windows 32 bit versions is 2000 MB This is for performance reasons Should you wish to write images to a CD easily for example this file limit can be changed to 700 MB for 80 minute CDs or 650 MB for 74 minute C
512. ull installation of iQ then go to Auxiliary Devices page Select Computer Boards DIO and DAC Control to install driver for the DAC card go through the installation by pressing next button default and select restart PC at the end of the installation 459 Andor Technology Andor iQ USER GUIDE y i TIA PNN Co AN DOR ANDOR iQ and Camera Drivers Installation ye TECHNOLOGY Installation Guide Software Protection Be ek on the links eee a Windows Utilities ee ANDOR iQ Software Camera Drivers O Data Translation DAC Control Kinetic Stage Control Oasis PCI Card O juxia Rages A Computer Boards DIO and DAC Control 4 Leica DMIRE DMRE Motorised Micrascope Control 4 Zeiss MTB Motorised Microscope Control Back To Start qu O Wa Selecting the Auxiliary Devices Options from the Installation CD shows the Options Choose Computer Boards and Follow the Instructions Device Manager Device Manager File Action View Help e meadar Fie Action View Help 4 4 D8K05G1J Computer O DAS Component Sea PCIM DDA06 16 Se Disk drives L Display adapters BS DYDICD ROM drives 4 Floppy disk controllers 2 Floppy disk drives i ve IDE ATAJATAPI controllers g IEEE 1394 Bus host controllers o DAS Component XO CI DIOZ4 See Disk drives 2 Display adapters 4 DYD CD ROM drives G24 Floppy d
513. uorescence transmitted and reflected paths respectively From the description above we can see that the transmitted and reflected signals can be approximated as follows T 0 5W C R 0 5 W C Eq 1 and simple algebra shows us that 113 Andor Technology Andor iQ USER GUIDE 2C T R W T R Eq 2 As Equation 2 highlights we need to collect both transmitted and reflected light signals images to compute the confocal signal Furthermore we can easily compute the wide field conventional epi fluorescence signal as well as the confocal The figure below shows how the T and R images are projected onto the CCD detector transmitted image T on the left and reflected image R on the right The simple mathematics of Equations 1 and 2 hide one complexity in the principle of the DSD i e the 7 and R images must be very well registered for the calculations to provide high quality images Any misalignment between the two will result in registration noise so an essential feature of image processing for the DSD is a high performance real time registration algorithm The DSD Calibration wizard delivers the parameters needed for the algorithm to be successful 3 3 2 DSD Calibration Wizard The DSD has an internal calibration mask which can be switched into the imaging path and used as a reference pattern to align the two image halves projected onto the image sensor The calibration mask pattern has a unique high contrast pattern t
514. urations for different scenarios and choose which one to use when you start iQ More details on the Configuration Manager are available in Appendix 2 In iQ 2 you cannot use Fast LZ mode But in order to maintain backward compatibility with iQ 1 X we have not removed the Fast LZ driver from the iQ folders so beware NOT to select the camera driver entitled Andor Camera with Fast Piezo Z and Monochromator a5 Device Configuration Manager File Edit Settings Tools About Exit Configuration Wizard New Edit Delete Exit Current Configurations op a Revolution FLZ Revolution Time Lapse a Y Created By Revolution Time Lapse Creator Not Found s te Date Created 04 11 2009 17 21 34 Date Last Modified 04 11 2009 17 21 34 Acquisition Device Andor Camera 7 Auxiliary Device 1 Andor Laser Combiner a Auxiliary Device 2 Andor FRAPPA Device a Auxiliary Device 3 Piezo Device a Auxiliary Device 4 Nikon TI Microscope a Auxiliary Device 5 Sigma KOKI Device a Auxiliary Device 6 Sutter Instruments Filter Device a Auxiliary Device Kinetic Trigger Device a Auxiliary Device 6 Yokogawa CSU Device oP A Auxiliary device OCX files used by this configuration Figure 4 1 1 Configuration Manager shows a Time Lapse Configuration Note that every device which is to be controlled must be listed in this Configuration The Configurations will control the same devi
515. urrent readout of the stage position RSS a ESES Lontral Fort ECT TAR AN Status Not Connected L Reset Hardwar The Hardware section is below Position in the e SE COA m motor control Settings Tab Check the box for each option being used stage control and or Z motor control as shown For each one selected a tab is added to the Setup Wizard page Use the Load and Save buttons to load or save a setting 482 Andor Technology Andor iQ USER GUIDE The Communications section allows you to select the Control Port from the drop down list If Reconnect At Start is selected the device is automatically recognized None Y when Andor iQ is opened None x Communication control Part COM2 kd Heconnect amp t Start Status Not Connected Reset The Status Section shows if the device is Connected and its location or if the device is Not Connected Status Not Connected Status Connected COMI The Reset button can be used to revert back to the original settings ee ee A3 7 2 The ASI XY Tab Enter the Device Resolution microns step for both X and Y The Default values are shown in the picture below PEITO Camera Ludi ASI LELITTTTTTTTTTTTTTTI Note The user must press Enter XY Stage Z Settings after changing the X value and again after changing the Y value Verify Device Resolution microns step that the Values entered were set by
516. urther information regarding Analysis Options We arrange the information in this sequence because Analysis and RGB Analysis share Data Options and so the information has been merged into a single section 254 Andor Technology Andor iQ USER GUIDE 5 4 RGB Analysis and Co Localization The RGB Analysis Tab opens the RGB Analysis Window which is used to calculate Statistics for color RGB images or for user defined regions on the image Acquisition Analysis RGE Analysis Calibration Movie Editor Spot Analysis E Settings Grey Level Statistics Channel Statistics Coincident Statistics Options 5 4 1 Grey Level Statistics Statistics are calculated for grey scale images Acquisition Analsis RGE Analysis Calibration Movie Editor Spot Analysis Analyse Analyse Settings Grey Level Statistics Channel Statistics Coincident Statistics Options Notes imago aga M22 Gre Min Thresh Grey Max Thresh Grey Agn to 10 00 m Oo 260 10 00 255 00 156 Pressing Analyse will perform the analysis on all regions defined on the image using the ROI tools if no regions have been defined the analysis will be performed on the entire image The results calculated depend upon the statistics selected on the Options page Section 5 5 1 and the threshold values applied in the Settings dialogue panel The data calculated from the analysis is displayed in the table Each row contains the statis
517. usting the alignment position size shape of the two rectangle ROIs in the Field Split E amp l 0 x Alignment ojja al ife melele wue 4m enro cR aw Load Save oK Close V Multi ESI venus if 7 Automap 100 229 394 11 wavelength Cho O Frame Number Min range Maxranc 4 z 4 gt fo fo fi Channel 1 Channel2 cia gt e oras Width 230 x1 12 x2 260 I Profile x xp 115 so ger 550 gez et Min et Ma a Hi bijejo i i ca ao Height 480 y fio y2 fio M Profley Yp 240 ha A e While the Field Split Dialog is showing and you adjust the ROIs the Main Image Window shows the two or four channel merged view This is a guide to course vertical and horizontal alignment For fine alignment you can use the Profile options which show the intensity profile in X and or Y across the selected points in the two or four channels The Field Split Window can be moved aside to see the new double channel image in the Image Window Select the Multi option to show both channels or select the coordinating box to show each individual channel 348 Andor Technology Andor iQ USER GUIDE 6 3 2 Field Split Window Toolbars and Options 7 1 x a Alignment e RJR pl Coe won ajo tows sme ox _ cose fines 7 Channel 1 gt Channel2 Width 230 x1 o x2 265 IV Profile x xp 55 fo an sso Height 490 ie v2 F P
518. ve C NMindor OD ataMehihar a Lab FL2 master Autolmages Acquistion Calibrator sexa O Nose Selected Su a Auoman ocx urson Acquisition Auxiliary Devices Pause Click DK to start protocol Interval O ma lastest DUGS7_BV 3877 Settings Experiment Exposure Sub Window Real EM Gan _ Andor iQ 1 10 2 Andor Camera Fe image ist Settings Q Wizards Pr EECA SHB umlole e t ol Window 512 512 I Enabled ES Andor Camera A ox Cuert Contest fe x ajajm gt mlolele tlol s Ro W User Ishihara Lab Administrato j Configuration TL slave a Image Focus2 Ancor jQ 14 bit grey pseudo 2 512 x 512 1 ion Default x1 Camera 1 easton Ansys AGB Analyse Golbraton Movie Editor Spot Anebas Status n ide sne Save Curent Auto Save F C Andor iOD ataishhara Lab TL lt tave Autolmages ia Acquistion Calibration Protocol nore 3 tae s Automap 1000 96505 uisttion Aux es Acquisition iliary Devi ary Devic Zz mon tite tyne INI ed r Camera DUBIZ BV 3248 Settings Exposure Sub Window Window 12 512 E a Sie PSTD Shutter 7 Open Close C Auto fix l E _ i 5 G Open C Close O Auto PreAmp Gan fio i Frames to be averaged Exposure Time I Frame Transter Use J p Frames to be averaged 1 50 00 ms T Ue KIM go
519. ve Image Save Display BMF F Save Display JPEG P Section 2 6 1 Print Open with Imaris This has the same functionality as the iQ File Menu Open with Imaris provides the same functionality as the main toolbar and launches Imaris with the currently loaded image The Save Display menu item includes the ability to save several different features of the image to disk in Bitmap or JPEG format Regions the positions of all user defined ROI s are saved Superimposed on a blank image Highlight all parts of the image highlighted by for example adjusting the threshold settings for an Analysis are saved superimposed on a blank image Image the source image is saved to disk All all contents of the Image Window are saved to disk including the original source image user defined ROIs and image overlays The Print item enables the contents of the Image Window to be printed using a standard Windows printer interface 71 Andor Technology Andor iQ USER GUIDE 2 0 2 The Edit Menu e Paula drosophila PBC_VerticalFlip_Rotate180 16 Bit 642 0 107 x 1020 0 107 4D File asia Image List Region View Fast MIP g Copy to Clipboard Nele olol s T ije TS a Edit Event Markers Add Event Marker Calibration Crop Image Dimension Editor Format Converter Selector 2 8 2 1 Copy to Clipboard The Copy to Clipboard item allows the contents of the Image Window to be copied and pasted
520. ware tightly synchronized Therefore the PCU is a key component to this module s functionality FLZ has 3 acquisition settings and the figure below shows the connections you need to make between the camera PCU and FRAPPA hardware to support these modes Revolution New FLZ and FRAPPA MPU PCU Connections 1 Standard FLZ in which the wavelength and Piezo devices are set and then the camera is triggered The simplest mode just requires that DO7 is connected to the Camera Ext Trig input 2 FLZ Fast Scan in which additional PCU TTL lines are used to sense the camera status Fire and Arm and change the wavelength and Piezo devices during camera readout to reduce settling delays In this case connect Arm to DI6 and Fire to DIZ 3 Frame Transfer mode where the camera overlaps readout and exposure to maximize performance In this mode settling times are not used and the camera Fire pulse is truncated to the vertical shift time Because of this we have programmed PCU output DO2 to be TTL 1 during the exposure So connect DO2 to an Active Blanking input for proper operation as shown in the figure above 552 Andor Technology Andor iQ How To Documents Note that we recommend you only run FLZ FT mode if you have a fast multi core processor to minimize software latencies which have an impact on exposure time Exposure time in this mode depends on the interval between trigger pulses so small variation in software timing
521. were in a physical film strip which if used for projection would show a movie Examples are shown below for both time series and Z series data The first file strip shows early embryonic development of a nematode C Elegans over a time period of a few tens of minutes with key frames chosen to illustrate a specific point The original 4D data was acquired with an Andor Revolution XD system and the result above shows a maximum intensity projection at each time point through the series The Strip View below shows a time series of cultured cells during a laser ablation experiment at the region defined by the red circle in the top left of the image frames A 350 nm 3ns 12 uJ pulse is delivered to the specimen between frames 29 and 30 When observed in a movie there is significant relaxation of local filaments and cellular junctions after ablation 303 Andor Technology Andor iQ USER GUIDE Strip View C DOCUMENTS AND SETTINGS M BROWNEWY DOCUMENTS ABD US CUSTOMERS UCL NANOWVP_ 2 EX Update Image Depth Dim Time 1 00 Draw Slices Snap O time O 00 00 026 Ta i a time 0 00 00 027 E time 0 00 00 030 ss time 0 00 00 031 E 5 10 1 Strip View Options Options Orientation fe ny xz zy x Size 64 512 pixels 192 Number of Columns 4 Slice Range 26 Increment i os W Slice Number VV Stamps inet View Width fi Colour Close Cancel The Film S
522. which depends on the exposure time range and bits per pixel of the camera Optimum results will generally be obtained by using Background correction with Greys per second 268 Andor Technology Andor iQ USER GUIDE 5 6 The Movie Editor Acquisition Analysis AGB Analysis Calibration Mowie Editar Spot Analysis Selected Image E 1 Path Default as gt 4 O 2 Animation dim Wavelength The Movie Editor is used to create Frame eee animation sequences from frames 7 a os E of interest in your images Which In Frame Rate i i WE can be saved as AVI Movies _in_ Out Ses ES Save List E Load List Load List Animation Dimension Wavelength Save Listas Movie Cees Seti eee poa Images included in the animation z Delete Selected Images Increment T arc displayed in the list at the O Multichannel ODEN rre Use Current Region C Play List bottom of the dialogue Save Overlay Image List DOM J os oe ees itn Saba Fo To add an image to the list either 1 Default 2 E favelength Defaukl 1 2 25 fale 1 Wavelength 1 Default 1 2 25 false 1 Wavelength 1 open it from the File Menu or Show it from the Image List To remove images from the list select them with the mouse and press Delete Selected Image Information on the selected image is displayed in the top right of the dialogue including the path image name animation dimension and number of frames There are two modes o
523. will be saved is C AndorBio imaging iQData User WhereUser is the User Name specified on starting iQ Note Even if Automatic Directory Selection is selected it is possible to select a different folder to which to save your data The data is saved in tab delimited format and can therefore be opened in Spreadsheet packages such as Microsoft Excel The x axis coordinates of the Analysis graph can be selected using the button to the left of the Threshold Toolbar Click on the button to toggle between pa On Frame number Pressing the Settings button opens a dialogue in which you can specify what intensities to consider Note In order to access the Settings button the Threshold option must be ticked in the Options tab DA7 O Andor Technology Andor iQ USER GUIDE L L Li Ee I l I I r I I I L l I I I p 1 E E E l e e e e E E i Li eo h e e e E E Li i l ee I i Overlay Colour Sere ees Use the Max and Min scrollbars to specify the minimum and maximum pixel intensity values that will be considered The specified limits are displayed in the graphs and the image displayed in the Image Window Is updated such that pixels included in the selected range are colored red thus enabling interactive optimization When the parameters in the Settings dialogue have been optimized press OK to close the dialogue accepting the specified values Pressing the Settings
524. with Grynkiewicz et al and Almers Neher calibration tools Key columns with G for Grynkiewicz and A for Almers Neher 395 Andor Technology Andor iQ USER GUIDE 6 12 Image lmage Math Plugin Mask Wizard Field Split Flip Rotate Image Image Filter Rolling Ball Filter Threshold Image Episodic Average ClearView Deconvolution Best Z Image Math Ratio Image Image Image Math Dual Image Disk The Image lmage Math Process is accessed from the Process Menu on the Control Window The process performs dyadic arithmetic operations between n Dim images which include addition subtraction multiplication and division between two source images to produce a destination EJE File About mM age E Source Background Mask Operation Add v Diadic Source Operate within N dimensional Image A RunningSUM1 Select selecting Math Image from the list opens the Diadic Math Window as shown below Operator Dimension Of Interest C Time The module provides several processing 7 a modes to be selected depending on the 4 Operate within N dimensional TIFF details of the Source Images These options C Operate on two images or image series Operate on two images or image series taking average of secondary C Self Operation taking average of selected range for secondary are listed at the bottom of the dialog and are Source Background explained in more detail below Threshold B 0 ic
525. x 22 um pixels running at 10MHz The Bl version provides peak QE of gt 90 IXon DU887 Fl front illuminated B back illuminated with 512 x 512 x 16 um pixels running at 1OMHz The B version provides peak QE of gt 90 IXon DU 885 with 1004 x 1002 x 8 um pixels running at 27 or 35 MHz These cameras use a front illuminated Virtual Phase CCD technology with peak QE 65 Luca S and R economical EMCCD range Clara high resolution 1 4 Mpixel cooled CCD camera When starting iQ the user will first be prompted to select the correct configuration You may then be prompted for a camera selection if so make sure to select the correct camera model In the future we intend to have multiple cameras under the control of a single iQ instance and then the serial number SN will be a suitable identifier The camera model and SN can be located on the back of the camera on the information sticker All cameras have similar controls in Andor iQ 446 Andor Technology Andor iQ USER GUIDE A3 2 1 Controlling the Andor iXon Cameras in iQ A3 2 1 1 Hardware Requirements To handle data transfer rates of 10MHz readout over extended kinetic series a powerful computer is recommended such as 2 4 GHz Pentium or Beiter 1 GB RAM and separate fast e g 10 000rom hard drive for Image Disk operations Also must be a PCl compatible computer where the PCI slot has bus master capability The computer must have an available auxiliary internal power conn
526. y observing the status of the respective Transmit and Receive messages 471 Andor Technology Andor iQ USER GUIDE A3 5 2 2 Z Acquisition Auxillary Devices 0 0000 P 472 Andor Technology Clicking on the Z tab displays the panel shown on the left This allows the resolution of the Z motor to be specified Andor iQ USER GUIDE A3 5 2 3 Settings Acquisition Auxillary Devices x Camera 0 ptizcan The Settings page is used to configure the Prior stage A Y Stade Settings Wisible Components For the example shown the current Shutter 7 MWheel A W Stage l Shutter WheelB Y Z motor devices in use are an XY stage and a Z motor The appropriate Communication Control Port com x Controller Type PRUSCAN g Status Connected on COMA checkboxes Stage and Z motor have been ticked for each device together with the checkbox Iv Reconnect At Start Reset Reconnect At Start This ensures Mae E i that the devices are recognised at W Report Manual Position PA ay a the start of the next iQ session 40 7 0 ZO RRE W Enable Joystick Receive 0 00 The stage is controlled through one of the serial ports on your computer From the Control Port dropdown list select the port that is being used for the control of the stage Similarly the Controller Type should be specified For the example above a ProScan controller is
527. y one device and the camera each device is accessed from a separate page tab If more devices are connected than can be viewed inscrolling arrows on the right of the page tabs are present use them to scroll through the devices The subsequent sections of Appendix 3 describe the setup and control of all hardware devices currently supported by Andor Technology 445 Andor Technology Andor iQ USER GUIDE A3 2 Andor iXon Andor is the innovator in electron multiplying EM CCD technology and first brought cameras utilizing EMCCD sensors to market in the shape of the iXon Range These cameras benefit from a patented permanent vacuum sealed detector housing unique single window design and world leading sensor cooling technology In a well ventilated laboratory the iXon cameras can maintain a stable sensor operating temperature or 70 C while chilled water provides 100 C capability Deep cooling of the sensor provides not only extraordinarily low dark current but also dramatically enhances EM gain EMCCD technology especially in the Andor iXon is leading the way to faster and more sensitive CCD detection with a significant impact in fields of scientific research as diverse as microscopic cellular imaging astronomy DNA sequencing spectroscopy and chemi luminescence The iXon family currently consists of 3 basic models with different pixel densities and EMCCD chips as follows IXon DU860 BI B back illuminated with 128 x 128
528. ytoplasm regions This is just an illustration of the tree and does not necessarily constitute a sensible experiment 567 Andor Technology Andor iQ How To Documents Andor iQ 2 2 Andor Camera oj xj File Image List Settings Wizards Process Analysis Plug Ins Help wa El amp a P Open Imaris Save Live Snap GFP Acquisition Analysis RGB Analysis Movie Editor Spot Analysis Status Auto Sav e G Andor iQD ata ndor ixon Prior 81 Piezo ALC Frappa Autolmages im Acquisition Calibratio El iXon 60Xoi Egy Protocol MyTemplate Protoco i 4 Image MyTemplate og Camera Binning Use Current Es Protocol oo Wait Allocation On Run we Channel 7 seen Camera Selection Use Current I XY Scan Events ap Repeat T 5 times 0 ms fastest 1 Repeat T1 10 times O ms fastest EM Repeat Channel BFP GFP x Channel Description ig Snap Frame oof End Channel iiti A Device Setup T Loop Protocol IV Display Image Show All ti End T1 af Frappa Frappa FRAPPA Selected Frappa Nuc ep Repeat T1 100 times O ms fastest EM Repeat Channel BFP GFP E Channel Description A Snap Frame M End Channel Ta Save As AG Options 2 Help il oe End 11 af Frappa Frappa FRAPPA Selected Frappa Cyto ep Repeat T1 1

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