Home

ScanR acquisition

1. 6 2 1 Well Plate Types 6 2 2 Stage Calibration Procedure 6 3 X Objective Parfocality Chapter 6 System Configuration 51 52 Chapter 6 System Configuration OLYMPUS 6 1 System Configuration Upon installation of the scan R screening station all hardware components have to be configured for the system to work properly The configuration has to be adjusted if changes are made for example if new filters are added of if an autoloader robot is to be integrated The execution of the System configuration command is password protected The System System Configuration command gives access to the Hardware configuration window with different tabs see following chapters Devices and ports to select the available hardware components and configure the PC ports Microscope to select objectives filters and contrast inserts MT20O to select the excitation filters of the fluorescence illumination system Error reporting Remote The System Stage Calibration command allows an absolute calibration of the stage with hard coded well plate types This is not to be confused with the stage initialization that is being carried out each time the software is started see Chapter 6 2 Stage Calibration 6 1 1 Devices and Ports YE System Configuration Devices and ports Microscope MT20 LED Error reporting Remote UCB COMI
2. 1 Execute System Objective Parfocality 2 Select the first objective to be used from the Objective short list e g UPLSAPO 20x It is convenient to start with the objective which has the highest magnification 3 Set the focus by using the Z focus wheel on the microscope and pressing Update when the sample is focused or by using the Z focus arrows slider 4 Make sure that the Parfocality value for the first objective is set to O Parfocality setup 1344x1024 0 5x 540 1314 262 5 Select the next objective from the Objective short list e g UPLSAPO 10x At first the image will be blurred 6 Set the focus for the second objective by using the up and down arrows in the Parfocality field 58 Chapter 6 System Configuration OLYM PUS Parfocality setup Karma Exposure Ume es 9 ro set Microscope Pier cube DAJH TR Objective UPC SAPO 10 Parfocalty um 12 gt Defined Color Channels fax 1344x1024 0 5X 407 1206 696 7 Repeat steps 5 and 6 for all other objectives for which parfocality is to be set The focus position will now be maintained when changing the objectives scan Acquisition Software Appendix The following chapter provides additional technical information 7 1 Data Storage Format sssssss 7 2 Error Message u tua Factory TOOS aut 7 3 1 LIGIPIGKUD EXE saei caesa Sis Grains exe dr land decd 723 2 Limo e
3. mas Lodi Fine Bb Bim cm Ixl PEEEEEPS Meroen Pie cube DATA D Dibieclkoe LAS LOS L Eos acum Cobre Chark nar Liita Q 5X a arcon The three positions are used to determine the Well diameter that will be set automatically in the Plate Type Settings window and the center position of well A1 The following dialog window will open OK Click here to repeat the measurement with the plate rotated by 180 This is required if an autoloader robot is used IF an autoloader robot is used the measurement has to be repeated with Ehe plate turned by 180 Press OF bo continue IF na autoloader robot is used click Cancel Cancel Click here if no autoloader robot is to be used In case of rectangular wells 1 Move a corner of the first well A1 to the red cross 2 Click Add Position 3 Repeat this for the opposite corner of the well the numbers field next to the Add Position button turns red to indicate that the measurement is completed 4 Confirm by clicking OK The two positions are used to determine the well height well width and the center position of well A1 scan Acquisition Software Chapter 5 Running a Scan 43 5 Running a Scan This chapter explains the steps that are necessary once a scan is started and the actions and online observations possible in the course of an experiment 5 1 Systems with Hardware Autofocus ccccccssseeeeeceeeseeeeeeeese
4. TimeFrame Argument number Examples CLIENT RunTo ExperimentEnd SERVER RunTo ExperimentEnd DONE CLIENT RunTo TimeFrame 6 SERVER RunTo TimeFrame 6 DENY Argument over range MoveTo Argument Argument might be one of the following PlatePickupPosition Only available before screen has started Moves to the Plate Pickup Position used for Robot and returns to last position when the next command is received PipettingPosition Only available after screen has started Moves the Plate by a fixed predefined XY offset and returns to last position when the next command is received The Pipetting offset position is defined in the utility FactorySettings LidPickup exe This can be useful for transmission light imaging Example of responses MoveTo PlatePickupPosition DONE MoveTo PlatePickupPosition DENY Screen is running MoveTo PipettingPosition DONE MoveTo PipettingPosition DENY No screen is running MoveTo PipettingPosition ERROR pipetting position cannot be reached for example Chapter 7 Appendix OLYM PUS 7 4 4 Known use cases and corresponding communication samples All settings files can be used in normal user mode as well Thus they can be tested and setup independently from the server client mode All data resulting from a client server mode screen is fully compatible and analyzable with the anlysis A scan hR is to be fully integrated into a plate sample preparation line 1 Test the different plates and assays in s
5. 3 3 Buttons Scan Buttons A click on the Start button starts the scan experiment using the current settings The Pause and Stop buttons will become active once a scan is started Settings Buttons The Edit and Open buttons lead to the interfaces needed for the setup of system parameters for a scan experiment Open Allows opening an existing experiment settings file By default the DefaultSettings xm file is loaded at startup This file is stored in the in the scan R installation folder in the directory AAcquisitionNScan Settings Library current user where current user corresponds to the Windows login name This means that when multiple Windows users start the system for each user scan H will load each user s default settings Scan Settings Online observation Pause Autofocus Settings lt Edit Allows modifying all the settings necessary for setting up a scan like autofocus acquisition settings and plate types see Chapter 4 Setting up a Scan Devices Can be used as a preview window and for changing defined color channels See Chapter 3 4 Devices Microscope Control and L ve View Online observation Buttons The Images Browse function enables you to browse through the already acquired images of the current or a previous scan It also allows you to delete entire color channel sets of images that shall not be considered in the analysis and to generate movies from time loop scans The Autofocus Settings function
6. Move the same well under the pipetting machine and press Set Pipetting position When the offsets are many centimeters large it will not be possible to move every well under the pipetting machine 64 Chapter 7 Appendix OLYMPUS 1s Lid Pickup Position Setup ATTENTION FACTORY CALIBRATION TOOL Inproper use might damage the System Joystick required for setup Please check Stage Lid collision whenever moving Stage Pickup position Adjust stage pickup position and press Set as Pickup Stage X um 20 0 Get Position Stage Y um me x um Y um Pickup Position 118625 0 t 738500 4 Pipetting position Move the objective to the center af a well and press Set well center Move this center af well under the pipetting machine and press Set Pipetting Position Set well center Set Pipetting Position x offset Lim Y offset um Pipetting affset 0 0 lag x 7 3 2 LimitSetup exe The options described in this chapter are only available with a motorized stage controlled by a STC controller With this program it is possible to define separate XYZ limits for each objective To activate or deactivate the limits in the Scan4R acquisition program check or uncheck the box Activate the limits in Scan4R acquisition If the stage is not calibrated run a stage calibration using scan 4R acquisition exe see Chapter 6 System Configuration The defaults might be used depe
7. Stage type RTC Serial COMS Autoloader Barcode reader Transmission shutter Z drift control ZDC Fast turret IX2 FRFACA scan Acquisition Software Chapter 6 System Configuration UCB Select the serial port RS232 of the imaging PC that is connected with the RS100 s universal control box UCB Stage Type Speed Click here to open the Stage speed window if you do not want to use the default settings for stage speed and acceleration Change the Velocity and the Acceleration using the respective sliders both for movements during a scan System Control and for movements under Joystick control TE Stage speed System control Joystick control Velocity 5 mm s Velocity 10 mrs m m is i v 100 100 Acceleration 100 mm s 2 Acceleration 95 41 mmjs 2 B v 100 100 sir acceleration Autoloader Check the box if an autoloader robot is connected o Note that the autoloader robot controller has to be connected to COM1 Barcode reader Check the box if a barcode reader is connected and select the respective serial port RS232 of the imaging PC Z drift control ZDC Check this box if a ZDC hardware autofocus device is connected Fast turret IX2 FRFACA If a fast filter turret is available this is automatically checked 6 1 2 Microscope Objectives Select an objective type from the shortlist for each occupied nosepiece position Filter cubes Set a filter cube
8. You can change the settings individually This is indicated by a behind the name of the color channel Click on save to store the new settings Illumination Fluorescence Transmission Select one of the two possible illumination modes epi fluorescence illumination with the MT20 or transmission illumination Illumination Excitation filter Contrast insert Select a MT20 excitation filter for a Fluorescence image or a transmission condenser contrast insert for a Transmission image respectively It has to match with the Microscope Filter cube selection Illumination Light intensity Use the slider to set the illumination intensity Focus Performs an autofocus procedure see Chapter 4 1 Autofocus Settings according to the current settings Ocular view Enables the eyepiece As long as the eyepiece is enabled the image is not updated and Ocular view enabled is shown in the upper left of the image Click again to disable the eyepiece Image display when ocular view is enabled 3 4 2 Z Focus Loarse Fine Step size ta ba Current position ca um E E Z focus Slider Use the slider for larger movements of the microscope Z drive scan Acquisition Software Chapter 3 The Main User Interface Z focus Coarse buttons Click on the arrows on the left to move the Z drive in steps 10 times as large as set in Step size Z focus Fine buttons Click on the arrows on the right to move the Z drive in steps as set in Step
9. 4 set of reference focus positions allows to interpolate the starting position for the software autofocus 1 Move to an arbitrary stage position 4 Repeat these steps several times 2 Focus the specimen 5 Click OK to confirm 3 Click Add to set the reference position Z focus XY navigation y Fine Step size 3 00 um Ez Current position J 5378 um Clipping Histogram W to reference list 0 Zmin um Zmax um Range um 4136 4226 90 1344x1024 0 54218 332 18 1 Click the Collect reference positions button to open the Reference Focus Positions live image window 2 Focus manually using the Z focus wheel on the microscope or use the Z focus tools see Chapter 3 4 2 Z Focus in the software to focus the sample You can use Ocular View to switch the microscope from camera view to ocular view When using the Z focus wheel on the microscope to set the focus do not forget to press Update to set this position in the software 3 Click the Add button to add the first position 4 Move the stage with the joystick or by using the XY navigation tool See Chapter 3 4 3 XY Navigation 28 Chapter 4 Setting up a Scan OLYM PUS 5 Repeat the Z focus measurement at two or more different stage positions that are reasonably far apart 6 From these measurements scan H will interpolate a grid of starting positions 7 At each measurement position the software autofocus takes the interpolated Z value a
10. Setting up a Scan 37 spacing between adjacent positions is set so that neither an image overlap nor a gap results It is calculated from objective magnification and camera chip size Pattern Set the number of Columns or Rows that are to be scanned within one well The shortlist contains also special patterns like single 2 2 full in and full out Positions in well Del key removes j selected positions Eu Full in Pattern Spacing Column spacing um Set here the distance between the centers of two adjacent Columns of positions Spacing Row spacing um Set here the distance between the centers of two adjacent Rows of positions Spacing Tile Click here to reset the spacing between columns and rows to the default without gaps or image overlap Spacing Field of view This is the specimen area covered by a single image It depends on objective magnification and camera chip size Spacing Total positions Shows the number of positions per well Positions in well oDer lke B The scheme shows the basic rectangular positions pattern relative to the entire well the green circle By default all positions will be scanned To modify this pattern use the tool buttons on the left to select a sub set of positions Remove the selected positions from the well pattern by pressing the Del key Full in pattern with removed central positions Selection Invert Click here to invert the
11. Type Gradient v Collect reference positions Set asstep width Repeat autofocus in each time lapse cycle Coarse autofocus Fine autofocus 8 T Layers 10 x Layers Camera Exposure time ms Binning v Step width um 2 81 Step width um 20 auto set 2x2 v w Microscope Scan range um Scan range um Objective Filter cube 100 45 307 1s UPLSAPO 10x DA Fi TR 807 60 Illumination 40 Fluorescence Su C Transmission Excitation Filter us Current position t Coarse focus position DAPI 350 50 20 Emission Filter 40 Filter 1 z 60 Intensity 50 1 e Object based coarse gradient fine This method combines the other methods to determine the focus It requires a ZDC hardware autofocus device The object based focus quantification is used for the coarse autofocus while the gradient based quantification is used for the fine autofocus o For ease of use it is recommended to select the Gradient mode Repeat autofocus in each time lapse cycle If the option is NOT active autofocus scans are carried out in the first cycle only The focal positions found in the first cycle are then used for the image acquisition in the following cycles If Repeat autofocus in each time lapse cycle is activated autofocus scans will be repeated in each time lapse cycle This reduces the overall speed of the experiment considerably but may be necessary if focus drifts are to be expected Chapter 4 Setting up a Sca
12. chip is composed of many light sensitive units pixels These pixels can be read out individually binning 1x1 or the signal of neighboring pixels can be combined electronically during data readout binning 1x1 Binning reduces the spatial resolution but increases the sensitivity and thus reduces the exposure time required for a good signal to noise ratio It further reduces the amount of data and consequently increases the readout speed Therefore binning is recommended if weak signals have to be detected at high acquisition rates or if spatial resolution is of minor importance 18 Chapter 3 The Main User Interface OLYM PUS Microscope Filter cube Select the filter cube to be used Note that a filter cube switch is time consuming even if the system features the fast IX2 FRFACA filter cube turret Imaging speed can be increased considerably by using multi band filter sets instead of several single band sets Microscope Objective Select the objective to be used Note that a switch of objectives during an experiment is possible but rather time consuming In order to maintain the same focus position for different objectives use the Objective Parfocality setup see Chapter 6 3 Objective Parfocality Defined Color Channels Select predefined color channels The objective filter cube illumination pathway excitation filter contrast inset and intensity of the illumination are set according to the settings of the predefined color channel
13. click position if possible while a Shift click will zoom out D Hand tool Hand tool Clicking this button in case of a zoomed in display enables you to move the visible area of the live image via mouse drag Image Display Status Bar The status bar below each display lists the image size the zoom factor and the intensity minimum and maximum 40h 4500 4000 1500 Jon 500 2500 5325 counts IDAPI je 2x512 0 5 283 ezzxSi2 05x 283 542 62 62 Display AF v i Aqisusul Display AF Check this box in order to display the images of the autofocus process in the bottom image display on the right side If also an image channel is selected in the drop down menu the display switches between the autofocus display and the selected image channel Note that there may be two different display settings set for the autofocus and the image display see Chapter 3 2 Display Context Chapter 3 The Main User Interface OLYM PUS Menu During the autofocus process the autofocus type Coarse Fine AF and the autofocus quality is shown in red in the upper left of the autofocus image System Status Bar The status bar at the bottom of the main window indicates the progress of the scan and gives an estimate for the remaining scan time It gives also the actual well number the total number of wells to be scanned the current position the total number of positions to be scanned the current time loop and the total number of tim
14. click to toggle wells on off Mouse drag Ctrl to select deselect wells pa eae Field of view um x um Edit selection table Wells selected 15 Md 867 x 660 Total positions Plate Tvpe test P Ed pte types Acquisition Well Descriptions Plate 24 Wel BD Falcon Well selection Well Mame Description Control A2 A3 Bl B2 p 200 w to Poe Selecting wells No wells are selected by default Click on a well to select it Click again to deselect it To select a group of wells drag a rectangle around the wells To deselect a group of wells press the Ctrl key and drag a rectangle around the wells Meander Wells Click here if the wells are to be scanned in meandering order rather than in numeric order row by row from left to right 4 4 2 Positions in well scan H is able to scan a series of positions within each well The pattern of positions to be scanned is defined in the Positions in well box on the right side of the Plate Manager To specify the pattern to be scanned within each well use the Positions Spacing and Selection fields The pattern is based on a basic rectangular pattern and can then be refined by manual selection of a sub set of positions The number of Columns and Rows as well as their distances can directly be entered in the corresponding fields Alternatively a special Pattern can be selected from the shortlist By default the scan Acquisition Software Chapter 4
15. e UCB not responding If the problem still exists after taking the measures described there contact your Olympus representative with a detailed description of the error message You entered at least one illegal character The e A file name input contained one of the listed characters File names characters V lt gt are not allowed may not contain any of these Cannot use the selected objective at this e XYZ limits are activated and the objective is not in an allowed position position Please move the stage to a secure XY position Please move the objective to a Z value inferior to um e Move the stage so that the objective appears in the center of the frame e Move the objective to a lower position if recommended 7 3 Factory Tools The programs described here are stored in the scan4R program folder in Aquisition FactoryTools 7 3 1 LidPickup exe Pick up position The Pickup position is used when performing a scan with an autoloader This is the position at which the stage will move when the autoloader adds or removes the plate Follow these steps to set up the pick up position 1 Move the stage to the desired pick up position 2 Press Set as Pickup Pipetting position The pipetting position is only used by the liquid handling interface see Chapter 7 4 Remote Contro Follow these steps to set up the pipetting position 1 Move the objective to the center of a well and press Set well center 2
16. hard disk during the scan Click the Test button on the Edit Scan Autofocus tab to open the Autofocus Settings window The functions needed to adjust the object recognition are shown in the Autofocus Settings window Threshold The threshold causes all image areas better said all image pixels of lower intensity to be ignored for object detection This prevents that background noise or low intensity artifacts are falsely detected as objects The value should be somewhat higher than the background intensity but lower than that of any cell or structure of interest Minimum cell size Activate this option if you want to set a minimal size for detected objects Enter the minimal number of pixels in the box below Maximum cell size Activate this option if you want to set a maximal size for detected objects Enter the maximal number of pixels in the box below scan Acquisition Software Chapter 4 Setting up a Scan 29 t Autofocus settings p Z Focus XY navigation L1 Coarse Fine Step size 3 00 um Current position J 5262 35 um 0 2000 44 Update 096 clipping Clipping 0 v mm Fine Z layers fine oup a ps o a g aN j Lu Lu 1 I Lu 1 Lu Lu l Lu gt m c in a e o o E X 1 1 1 I 1 1 1 I I 1 1 OSs 2s 4 BS POUR cA3Coarse Z ayers coarse Ocular view 672x512 1X 233 538 1 Objects gt i Minimum cell size Maximum cell size Mark center Frame object Threshold 280 36
17. holder at the same time is only supported if manual loading is chosen e You have not taught a starting position for the scan Please choose yes if you are asked to teach a starting position after starting a scan 62 Chapter 7 Appendix OLYM PUS No stage pick up position has been defined A scan is configured with an autoloader but the stage pick up for the robot To define it please exit the position for the robot was not yet defined software and use the tool FactorySettings LidPickup exe Define the pick up position with the utility LidPickup exe Scan cannot be started Not enough space left to fulfill your request The hard disk space left will be not large enough for the scan For robot loading a number of 20 plates is assumed The remaining space is sufficient for complete plates repetitions Free hard disk space or change the configuration of the scan Warning images of one or many At the start of the screen it is checked if all subpositions will be inside subposition s will not be taken because they the limits when XYZ limits are activated are outside the XY limits for the selected objective Check if the plate is right calibrated in Edit Plate manager Check if the subposition pattern is inside the well Check the XY limits for the screening objective in Devices XY navigation limits are drawn as white lines Image browser Error Message Explanation Possible Measures An error occurre
18. list of the Acquisition tab is shown The quality of the autofocus is shown as a graph 6 In order to change the color channel the illumination or microscope settings you can move the Autofocus settings window aside to have access to the Edit scan Acquisition tab The changes made in the Acquisition tab will immediately be applied o When opening the Live settings control window the Acquisition tab remains active Move the Acquisition settings windows aside to get access to the Acquisition tab and to change the settings e g color channels exposure time etc You can now change and check the settings in the Edit scan Acquisition tab and have control over these settings in the Live settings control window simultaneously The status bar below the live image display gives information about the image size the display zoom factor the image type as well as the intensity and the coordinates of the cursor Check the exposure time for all Color channels and avoid over saturating the camera by keeping the maximum intensity well below 4095 counts the maximum of a 12 bit camera Adjust the Camera Exposure time on the Edit Scan Acquisition tab accordingly Ti Live settings control P Z focus XY navigation Z offset relative to the AF position Coarse Fine Step size 3 00 Z offset 0 00 um Update um Clipping Histogram W Fine Z layers fine gt u m m c un u o o 24 I lt
19. o The burner will be ignited automatically after system and software startup It is recommended to allow a warm up time of 10 min to let the light output stabilize before starting the first screening run Start the screen by clicking the Start button on the main window The following steps depend on the available hardware e f a ZDC hardware autofocus is available and standard plates with a defined position of well A1 are used scan4R will carry out the scan without further user input Scan Acquisition Software Chapter 2 Short Step by Step Guide 9 e f no ZDC hardware autofocus is available scan R requires the user to set interactively a starting Z position for the software autofocus Afterwards the scan will be carried out automatically e f spotted slides or other sample holders without defined starting positions are used scan R requires the user to move interactively to the center of the first well If an autoloader robot is to be used the scan will be performed on all plates on the stackers 1 and 2 Afterwards the plates will be returned from stacker 3 to their original positions o The autoloader robot is not able to identify the plate thickness If you change the well plate type you will probably also have to change the robot method Note that loading well plates with or without cover also affects the effective stacking thickness of the plates this has to be taken care of in the robot settings Do not remove the posit
20. of the software can only be made in compiled form as part of the software developed by the User under strict observation of the conditions set forth in the written permission to the User The User must include the OSIS scan R SOFTWARE PRODUCT copyright notification with the User s software The User has to make sure that OLYMPUS SOFT IMAGING SOLUTIONS cannot be held liable for any damages or injuries resulting from the use of the User s software that include parts of the OSIS scan R SOFTWARE PRODUCT 4 Copyright 1 OLYMPUS SOFT IMAGING SOLUTIONS or its subsidiaries remain owners of the software and its documentation With the purchase the User obtains ownership of the diskettes or other physical storage OLYMPUS devices excluding the software and other data contained thereon and the manuals 2 OLYMPUS SOFT IMAGING SOLUTIONS reserves the right to all publications duplication editing and marketing of the software and the software documentation Without prior written permission the User may not change translate de compile or de assemble the software copy any of the written or printed documentation of the software rent lease or license the software to a third party 3 The license property and user rights to the OLYMPUS SOFT IMAGING SOLUTIONS software disks and manuals may only be sold or transferred to a third party on a permanent basis if the third party agrees to abide by the conditions in this co
21. or an online view of the auto focus procedure Display AF BSP very E DAPI vj NONE v fosxoo0 2 310 The drop down menu below the large image display allows selecting NONE single channel or a RGB Overlay transmission to be displayed in the main panel When RGB Overlay is selected the drop down menus in the colored boxes become available The color channels to be displayed in red green blue and gray can be selected here A transmission channel can be selected in the gray drop down list scan Acquisition Software Chapter 3 The Main User Interface 13 The clipping set for the single image channels will be used for the RGB Overlay Note however that the colors may differ from the single image channels depending on the colors selected there cf Chapter 3 2 Display Context Menu When RGB Overlay is selected in the main image the image status bar lists the zoom factor the intensities in each color channel as 8 bit value and the X Y pixel value of the current mouse pointer position When a single color channel is selected in the main image the image status bar lists the zoom factor the intensity as 12 bit value and the X Y pixel value of the current mouse pointer position 1344x1024 0 25 265 816 228 Display AF Color channels Select the color channels from the shortlist underneath each display Zoom tool Zoom Activate the tool and click on a display to zoom into the image by a factor of 2 centering on the
22. pattern of selected positions Selection Reset position defaults Click here to return to the basic positions pattern as defined by Columns Well and Rows Well and the default autofocus settings for each position see the Selection context menu 38 Chapter 4 Setting up a Scan OLYMPUS Selection Fill Click here to fill a ROI e g a drawn circle or rectangle with sub positions Selection context menu A right click on a position opens its context menu This context menu allows selecting the kind of autofocus to be carried out at every position individually Depending on the sample it may or may not be useful to carry out the hardware autofocus and or the coarse software autofocus only at the first position and only the fine software autofocus at each position w AF Fine w AF Coarse w AF Hardware Acquisition Meander Click here if the positions are to be scanned in meandering order rather than in numeric order row by row from left to right Acquisition Center Start For scan patterns full in and full out the central position is selected as starting position Acquisition Show order Click here to display an animation of the optimized position acquisition order Click again to stop the animation 4 5 Plate Type Settings T Plate Type Settings 24 Well COSTAR 3524 Type 48 Well test 41 position calibrated 48 Well COSTAR 3548 96 Well COSTAR 3596 6 Well NUNC 140675 12 Well NUNC 150628 Patter
23. selected color palette Percentage values can be set between 096 and 5 1 is usually a good value to start with a slight increase in contrast and brightness results If the value is set too high the dynamic range is reduced visibly and the image may look oversaturated Note that if you use 96 clipping the intensities of different images will be scaled separately such that you might not be able to visually compare the intensities In order to get equally scaled intensities for all images use the histogram clipping T Display Settings Scaling color Ec Click to choose color NEN L HBBEBBBBHBEBEHEBBEBEBEE History System B DD Op ud in cm EN Qs t Display Settings 3500 auem Scaling color BEEN Click to choose color 00 zy m 2000 ud X Intensity clipping o 1000 300 counts The histogram on the right side of the image displays shows the intensity distribution in the main display only in case of single channel displays If the option Histogram is selected in Intensity clipping green horizontal lines that signify the clipping limits can be mouse dragged to adjust the maximum and minimum intensity clipping and thus to optimize the image contrast Note that the display values are adjusted for the channels This means that if you change the channels the display settings are changed according to the settings last time the channel was used 16 Chapter 3 The Main User Interface OLYM PUS
24. size Z focus Z drive steps um Set here the step size for the z drive when using the z drive buttons Z focus Update Click here to update the Current position after moving the Z drive manually at the microscope 3 4 3 XY Navigation focus XY navigation Row Column Use these boxes to move the stage to the well of interest Make sure that the correct well plate type is selected and that the stage is calibrated See Chapter 6 2 Stage Calibration Navigation Interactive Scheme The blue circle represents the well currently placed under the objective The white rectangle represents the field of view of the camera Click anywhere in the scheme and stage will move this spot to the center of the field of view When XYZ Limits cf Chapter 7 3 2 LimitSetup exe is activated white lines represent the limits of navigation for X and Y 3 5 Menus File Open This command loads scan settings from experiment descriptor xml files It can also be executed with the Open button File Save as This command saves the current scan settings to an experiment descriptor xrml file Note that this is performed automatically upon starting the scan File Edit This command allows to edit the current scan settings and to define new types of formats see Chapter 4 Setting up a Scan It can also be executed with the Edit button File Exit This command shuts down the scan H software View Status Bar This command minimizes the scan R wi
25. the main panel and on the graph on the right the quality of the AF procedure is displayed The focusing was successful when both curves for fine and coarse AF show a pronounced peak To optimize coarse and fine range change the corresponding settings in the Autofocus tab and click again Focus in the Autofocus settings window to run the AF procedure You can also navigate to other positions on the plate using the XY navigation tool see Chapter 3 4 3 XY Navigation When you are satisfied with the result close the Autofocus settings window by clicking OK scan Acquisition Software Chapter 4 Setting up a Scan 27 4 1 4 Autofocus with Interpolated Starting Positions Step by step This procedure may be helpful if no ZDC hardware autofocus is available If the focus positions at different stage positions differ strongly it is likely that the software autofocus will not be successful unless information about a suitable starting position is available Therefore it is possible to detect focus positions at different stage positions first and then to interpolate these positions to obtain a plane of starting positions for the coarse fine autofocus Interpolate AF start positions Activate this option on the Edit Scan Autofocus tab to enable the interpolation Collect reference positions This button opens the Reference Focus Positions window where up to 40 reference positions can be set LT 14 14 ir 2d Reference Focus Positions _
26. um 5 Set the Coarse autofocus Step size to 10 um It should not be larger than half the Fine autofocus Scan range 6 Set the Coarse autofocus Scan range to 50 um for example 26 Chapter 4 T 8 10 Vie 12 13 Setting up a Scan OLYM PUS Once finished click the Test button The Autofocus settings live image window will open and display a live image acquired with the settings of the Autofocus tab Focus manually using the Z focus wheel on the microscope or use the Z focus tools See Chapter 3 4 2 Z Focus in the software to focus the sample roughly You can use Ocular View to switch the microscope from camera view to ocular view When using the z focus wheel on the microscope to set the focus do not forget to press Update to set this position in the software fc Autofocus settings p 2 focus XY navigation Fine cM um Current position J 5262 35 um o 0 2500 5150 Update 0 clipping Clipping 0 v Z layers fine 6 Autofocus quality mE I I I I I 1j ps2 sag pL Bg S9 c3Coarse Z layers coarse In order to change the illumination or microscope settings you can move the Autofocus settings window aside to have access to the Edit scan Autofocus tab The changes made in the Autofocus tab will immediately be applied Click Focus to run the autofocus First the coarse AF is performed and afterwards the fine AF Afterwards the focused image is displayed in
27. upon software start and thus does not have to be determined prior to the start of a scan Calibrated well plates are highlighted green in the Plate Manager Plate Tvpe Tvpe 48 Well best A1 position calibrated c Non calibrated well plates Before the start of a scan the user is asked to move the stage to the A1 position Non calibrated well plates are not highlighted in the Plate Manager Plate Type a6 well v Edi plate types Type 12 wall 41 position uncalibrated 6 2 2 Stage Calibration Procedure Corner selection for calibration Ti Please choose Plate for System Ca E Please select the stage Frame corner which will be calibrated left right up Corner har position Well plate type EB bal Corner verb position The calibrated corner is at the opposite side of the hook 1 Place a system standard well plate on the stage and select the well plate type in the Plate Format window scan Acquisition Software Chapter 6 System Configuration 57 2 Execute System Stage Calibration 3 Perform the 3 point A1 Measurement as described in Chapter 4 5 1 Once the procedure is completed the stage is calibrated and the A1 position of system standard as well as calibrated well plates will always be located at a known position 6 3 Objective Parfocality This command allows you to set an offset for the Z position for the used objectives so that when changing the objective the focus is maintained
28. value Illumination Fluorescence Transmission Select one of the two possible illumination modes epi fluorescence illumination with the MT20 or transmission illumination Illumination Excitation filter Contrast insert Select a MT20 excitation filter for a Fluorescence image or a transmission condenser contrast insert for a Transmission image respectively It has to match with the Microscope Filter cube selection Illumination Light intensity Use the slider to set the illumination intensity of the MT20 and the transmission light respectively Z stack settings Layers Set the number of layers of the Z stack Z stack settings Step width um Set the distance between the layers of the Z stack Z stack settings Projections If this option is checked a Z stack will be acquired but not saved as such The software calculates a maximum intensity projection fitc_zMax tif a maximum minus mean intensity projection from Z stack fitc_zMaxBkg tif and a ayer of origin image of the maximum intensity projection fitc zMaxZ tif and only keeps the central layer i e the layer found by the AF procedure of the original stack fitc tif All four images are then being stored as a four channel image Recommended to use with high Numerical Aperture NA objectives use zMaxBKQG tif for object segmentation in the Analysis fitc tif fitc_zMax tif Eua ju eL fitc zMaxBkg tif fite zMaxz tif scan Acquisition Software Chapte
29. 1 l I I nn 33 pb Cf c3Coarse Z layers coarse Ocular view 1344x1024 D 5x274 382 14 OK TUER o The camera has a bit depth of 12 so that saturation is reached at pixel values of 4095 However the pictures are stored with 16 bit depth for reasons of computer efficiency scan Acquisition Software Chapter 4 Setting up a Scan Z offset relative to the AF position Use the arrow buttons to determine the focus offset of the different channels relative to the position found by the autofocus The offset is useful for imaging structures that are located in a different Z position within the sample than the position found by the autofocus The Z offset set in the Live settings control window is also entered in the Z offset field of the Edit Scan Acquisition tab Color channels are switched by selection on the Edit Scan Acquisition tab Note that in the Live settings control window you can only change the Z offset but not the Autofocus properties Autofocus quality Coarse and Fine These two graphs enable you to judge the performance of the software autofocus If either graph shows a pronounced peak the focus has been found and the settings in the Edit Scan Autofocus tab are suitable If the maximum of one or both curves is located at the left or right border of the scan range the settings have to be adjusted o If the Autofocus quality functions do not show a pronounced peak the focus has likely not been found Enlarge
30. F plane It is required to make time lapse images of the same position before and after the insertion to observe the changes due to the insertion Example 1 In the scan R Acquisition software execute Edit scan Acquisition and select Loop Well in the Time lapse settings box and adjust the settings Only one subposition should be selected for the settings 2 Communicate with the server like the following workflow 2 wells have been selected in the scan4R acquisition GUI the number of time lapse frame has been set to 40 RunToTimeFrame 20 DONE 12 Chapter 7 Appendix OLYM PUS Tr 00000 S an Armee 000 S Ka 00000000 Fressen ron 00 EMEN RunTo Well XYZ RunTo Well XY ERROR RunTo Well XY not reached until end of experiment RunTo ExperimentEnd DENY No screen is running a zb Hemote control Commands always available Quit GetCominterfaceVersion Gethumber Wells GetNumber Subpositions Gethumber Timetrames Commands available before screen StartExperiment Experiment path relative to Scan Settings Library SetExperimentCluster test2 xm MoveTo PlatePickupPosition Commands available during screen RunTa ExperimentEnd RunTa PlateEnd RunTa Well xY RunTo Well XYz RunTo Subposition XY RunTa Subposition 72 Timetrame value RunTa Timeframe D MoveTa PFipettingPasitian StopExperiment Command SetExperimentCluster test2 xmll Done Value 3 T Deny E Errar Scan Acquisit
31. LION ee 1 1 1 Abstracts c E 2 1 2 TEchnicalSuUppOtlssse ee ee 3 INE i o 3 2 Short Step by Step Guide 0z42200200000000000000n0nnnnnnnnnnnnnnnn Rare nn nnns 5 Jj Main User Interface seele 11 294 urobbPariel Displays arnan eon ec 12 3 2 Display Context Men ciet udi reelle ee 14 3 3 IURE RN 16 3 4 Devices Microscope Control and Live ViCW ccccccceseeeeeeeeeeeeeeeeeeeeeeeess 17 34 1 MIGIOSCODO SOLOS su ke ehe 17 AD SEIEOCUS ee ee een C LER 18 AI IRV INAVIG ATION ota eect auto a ticket ot aou dua ratae a aucta eek 19 SEM ITUR EU 19 4 Selling up ad Scan 22 oe vehere di va teu ee ee 21 AT AUIOIGCUS SCHINGS zus 22 Adsl Coarse AULOIOCUS sc c 24 4 12 sPING Piensa cc c 25 4 1 3 Gradient Autofocus Adjustment Step by step sss 25 4 1 4 Autofocus with Interpolated Starting Positions Step by step 27 4 1 5 Object based Autofocus cccccccccsssseceeceeseeeecceeeseeeecseaseeesesauseesesseaees 28 4 2 Acquisition Settings ccceeeeeececeeccceesseeeeeeecceeeuseeeeeeseeseaaaseeeeeeeessaasseeeeeess 30 43 Bve 6 EPA PvE 33 4 4 Plate Managers ei ne Be een 35 Al NV CMO AUC iit Eee an use 35 454 2 POSIUONSIN WO en aan naeh 36 4 5 Plate Type Sells daas nti ee ee 38 AO well Measurement en sweet 40 5 RUNG A SCAN ea ern 43 5 1 Systems with Hardware Autofocus uuuusssennnnnennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
32. OLYMPUS INSTRUCTIONS scan AUTOMATED IMAGE ACQUISITION SOFTWARE This instruction manual describes Olympus scan Automated Image Acquisition Software for Life Science To ensure safety obtain optimum performance and familiarize yourself fully with the use of these products we recommend that you study this manual thoroughly before operation Together with this manual please also read the scan Automated Image Acquisition Hardware manual as well as the manuals of all other devices controlled by this software in order to understand general operation methods Retain this manual in an easily accessible place near a system for future reference OLYMPUS SOFT IMAGING SOLUTIONS GMBH Rupert Mayer Strasse 44 D 81379 M nchen Tel 49 89 89 55 805 660 Fax 49 89 89 55 805 6606 Email info olympus sis com www olympus sis com OLYMPUS Imaging Excellence We at Olympus Soft Imaging Solutions GmbH have tried to make the information in this manual as accurate and reliable as possible Nevertheless Olympus Soft Imaging Solutions GmbH disclaims any warranty of any kind whether expressed or implied as to any matter whatsoever relating to this manual including without limitation the merchantability or fitness for any particular purpose Olympus Soft Imaging Solutions GmbH will from time to time revise the product described in this manual and reserves the right to make such changes without obligation to notify the purchaser I
33. Plate Manager window will open Enter a name in the Plate name box If the plate name remains unchanged in the course of consecutive scans the scan directories will be stored with an incremented numeration appended to the name Select the well plate format from the Format shortlist T 8 Chapter 2 Short Step by Step Guide Tis Edit Scan ee Autofocus Acquisition Plate Manager well pattern scan skip Left click to toggle wells onjofF Mouse drag Ctrl to select deselect wells Meander wels Well diameter um 6349 Positions in well Del key removes selected positions Pattern Columns 3 Rows i2 m Specific e Acquisition Center start Meander n Spacing OLYMPUS Column spacing urn leery E Row spacing um 6 Field of view um x um 1867 x 660 Edit selection table Plate Type Total positions 96 Well B Edit plate types 3 OK Cancel Select wells for acquisition by clicking on the circles that represent the wells Deselect by clicking again Dragging the mouse over a range of circles causes multiple selections Deselect wells by dragging the mouse while pressing the Ctrl key Click Edit selection table to get a list of the selected well numbers and their Names or Descriptions The names can be changed at will Confirm the settings and exit the Edit Scan dialog by clicking OK Start the Screen
34. a ZDC hardware autofocus is available the system will first perform a complete hardware and software autofocus run as defined in the Edit Scan Autofocus tab see Chapter 4 1 The Autofocus Settings When the AF procedure is completed the Live settings window opens 2 If no ZDC is available the starting position for the software Autofocus has to be set first In this case the Manual Focus window opens The settings of the Autofocus tab are used for this display i e the color channel used for Autofocus is shown ES Manual focus The software autofocus requires a starting position 1 Focus the specimen 2 Ck OX to corr Zfoos XY navigation M r 5x ar E Lose fre ld do p o 5 ea Current position 3 24 um m IFFI Update 3431004 0 5 215 64 8 33 34 Chapter 4 Setting up a Scan OLYM PUS 3 Focus manually using the Z focus wheel on the microscope or use the Z focus tools see Chapter 3 4 2 Z Focus in the software to focus the sample You can use Ocular View to switch the microscope from camera view to ocular view When using the Z focus wheel on the microscope to set the focus do not forget to press Update to set this position in the software 4 Click OK to start the software autofocus 5 Once the autofocus run is completed the Live Settings window will appear in addition to the Edit Scan window The color channel selected in the Selected Color Channels
35. allows adjusting the autofocus settings during a running scan if necessary see Chapter 4 1 Autofocus Settings scan Acquisition Software Chapter 3 The Main User Interface 17 3 4 Devices Microscope Control and Live View The Devices button opens the Device Control dialog that starts live image acquisition and allows to access a selection of hardware settings directly The device view can be used as a preview to get an impression of the sample and to change defined color channels Tie Devion control Expasurs lime ine a set Carrara Enea A 427 155 2 3 4 1 Microscope Settings Camera Exposure time ms The exposure time determines the period of time during which the CCD chip is sensitive to incoming light in other words during which photons are collected and converted into charges to be read out afterwards Enter a value manually or use the Auto set button Auto set Use the auto set button to obtain a value for the exposure time The exposure time is set such that the mean intensity of the objects equals a certain value When the original image was saturated it may be necessary to press the several times in order to set the exposure time properly Note that when using the Auto set button in order to set the exposure time for different assays the value set for the intensity threshold object detection algorithm of the scan R Analysis software will be the same for these assays Camera Binning The CCD
36. ated by a red box 4 2 Acquisition Settings Selected Color Channels list This lists all currently defined color channels in the order of acquisition Select a channel by mouse click for modification Drag a channel up or down to alter the channel acquisition order New Click here to add a new color channel to the list The new channel will be given the name Blank by default and can be changed in the Name box It will be used for the image file name upon storage Remove Removes a channel from the selected color channels The channel will be stored in the Defined Color Channels list Defined Color Channels List of predefined color channels The predefined color channels can be changed in the Device window Settings Devices Add to list Adds a selected color channel from the Defined Color Channels to the Selected Color Channels The color channel will be added at the bottom of the list Delete Click here to delete the listed Image type from the Defined Color Channels list scan Acquisition Software Chapter 4 Setting up a Scan Live settings Click here to start a live image acquisition in a new window that opens This allows observing the influence of the settings changes online See Chapter 4 3 Live Settings Note that first an autofocus scan will be carried out When no ZDC is available the sample a starting position has to be selected for the autofocus procedure After that an autofocus is performed and the Live settings cont
37. can Screening Station for Life Sciences are for research use only 1 1 Abstract This user manual will guide you through the usage of the Olympus Screening Station and will assist you in setting up efficient and reliable screens The Screening System scan H is intended to be used for the screening of well plates and slides Do not use the system in other ways than described in this manual For detailed information on the separate system hardware components please refer to the corresponding manuals delivered with the system The Screening Station scan R consists of the following hardware components e cellAR Imaging Workstation hardware only e X81 microscope e Microscope stage e ZDC hardware auto focus device optional e Autoloader robot optional e Laboratory workbench optional Please keep in mind that all the components are designed to work together as an integrated system Taking away or replacing single components without prior consultation of Olympus Soft Imaging Solutions is strongly discouraged and will most probably impair the performance of the system dramatically Any damages to the system due to such mishandling will not be covered by the guarantee All necessary tools for handling and alignment are shipped with the system To ensure safety and optimum performance as well as to familiarize yourself with the use of the system we recommend that you study this manual and the hardware component manuals thoroughly b
38. can H Save the different setting files in the directory Scan Settings Library Every kind of scan available in the user mode will also be available in the server mode 2 Communicate with the server according to the following workflow Be pe MEE SetExperimentCluster PrefinedExperiment1 SetExperimentCluster DONE ire rte 0 LILLCLLLL MM NNNM mn 000 SetExperimentCluster PrefinedExperiment2 3 Analyze separately each scan in scan R analysis The analysis has to be run in batch mode or in parallel The batch has to be fed manually B Well Time lapse acquisition using a liquid handling automate trigger substance for starting the time lapse Analogous for Subpositions or Plate Time lapse The liquid insertion changes the AF layer position Therefore AF should be performed after liquid insertion Example 1 In the scan R Acquisition software execute Edit scan Acquisition and select Loop Well in the Time lapse settings box and adjust the settings 2 Communicate with the server like the following workflow 3 wells have been selected in the scan4R acquisition GUI Scan Acquisition Software Chapter 7 Appendix 71 rts C ive RunTo Well XY LLC 700000007 retraite BEEN LL ANN RunTo Well XY DONE EE pe S a 00000000 C Well Time lapse acquisition using a liquid handling automate trigger substance for starting the time lapse The trigger point is time sensitive and insertion of the liquid does not change the A
39. d during deletion Please e his error message may appear when you delete files while an check that you have the right permissions analysis routine is running Make sure that no external program such and no other program locks the data as an image file viewer has opened any of the files to be selected Loading a scan File not found e Choose an existing file You are trying to load a new XML file with an e Update the software to load the scan settings older software version Please update Error occurred while loading scan settings e The file was not a correct settings file Miscellaneous dcam wait e Atimeout occurred during camera operation that is an image could not be taken in time during the scan Make sure that no other software blocks such a big part of the system resources that the image acquisition is disturbed For further operation restart scan H Check that the camera trigger cable is connected properly For further operation restart scan4R Stage Position not reached within time limit e This error may occur during the stage calibration Repeat the Please check your connections and settings calibration in the following manner Exit scan R Reset the stage controller by switching the power off and then on again Move the stage with the joystick so that the stage insert is centered above the objective Restart scan HR Scan Acquisition Software Chapter 7 Appendix 63 e Stage not responding
40. e It is not recommended to activate this switch if expected idle time of the system is less than an hour Excitation filter Set a filter name for each occupied position in the MT20 filter wheel 6 1 4 Error Reporting Remote Error reporting Error reporting Check this box if you want error messages that may be generated during a scan to be sent automatically as emails Email address and SMTP Make sure to set the correct addresses here Subject and Body You may enter default content here to be sent in addition to the error message Remote The information here is only needed to allow communication between the scan H analysis software and the system from a remote PC Make sure that remote access is enabled in the corresponding MS Windows settings scan Acquisition Software Chapter 6 System Configuration 55 YE System Configuration YE System Configuration Devices and ports Microscope MT20 LED Error reporting Remote Devices and ports Microscope MT20 LED Error reporting Remote Error reporting Email address youradress domain org Allowed hosts 10 2 11 178 localhost Subject Subject of mail Body Write your own message down here SMTP server 10 2 11 62 Port and Allowed hosts Make sure to set the correct addresses here to allow remote access to the system 6 2 Stage Calibration When the scan4R Acquisition software is started the stage initializes automatically to determine its maximu
41. e focus do not forget to press Update to set this position in the software 30 Chapter 4 10 11 12 Setting up a Scan OLYM PUS Set a value for Threshold To determine a meaningful value activate the pointer tool by clicking the arrow button in the tool bar at the bottom right side of the image window Move the cursor over a background area towards the border of an object The image information bar below the image window list the intensity at that point For an initial try you may set the threshold to such a borderline value Activate Minimum cell size and click the ROI and draw a rectangle which is somewhat smaller than the smallest object to be detected Click the Set current ROI below Minimum cell size The value is entered in the corresponding pixel field Alternatively you can directly enter a value in the pixel field Activate Maximum cell size and click the ROI and draw a rectangle which is somewhat larger than the largest object to be detected Click Set current ROI below Maximum cell size The value is entered in the corresponding pixel field Alternatively you can directly enter a value in the pixel field Check Mark center and Frame object Click Focus to run the autofocus First the coarse AF is performed and afterwards the fine AF Afterwards the focused image is displayed in the main panel and on the graph on the right the quality of the AF procedure is displayed The detected objects are numbered and indic
42. e loops to be performed as well as the current plate number and the total number of plates to be measured The MT20 icon indicates if the burner of the MT20 is switched on or off Well 2 of 16 Position 1 F of 9 Time 1 of 2 Plate 1 of 1 Time remaining 00 30 himin MT20 n Progress M 3 2 Display Context Menu Palette Grayscale Display Settings Binary Gradient Rainbow Temperature User Defined A right click on a display opens a context menu that allows to select additional display settings and to configure the multicolor representation Palette Use this command to select the display mode for single channel displays Display Settings Use this command to setup the color display mode It opens the Display Settings dialog Display Settings Scaling color a Click Eo choose color Intensity clipping Display Settings Scaling color Click on the Scaling color box to open a new window that allows you to choose a new image display color Note that this color is not used for the RGB overlay Display Settings Intensity clipping To make automatic scaling more effective when noise or bright impurities are present you can define how many pixels as a percentage of the total number of pixels scan Acquisition Software Chapter 3 The Main User Interface will be displayed uniformly with maximum or minimum brightness The intensity of the remaining range of pixels will then be scaled linearly according to the
43. e scan R acquisition software by double clicking the scan R Acquisition icon on the desktop fr Checking hardware components Checking hardware components Please wait Upon start the software will check the availability and status of all hardware components and inform you if one of the components is not available In that case switch on the missing device or configure the ports as described in Chapter 6 System Configuration and execute the command Settings Initialize Devices in order to initialize all devices without starting the software anew The software will inform you that it is going to calibrate the stage and ask you to confirm Remove any obstacles from the stage s operating range and press the Yes button The stage calibration will take a few seconds Your stage will now be calibrated Please make sure that Ehe whole travel range is Freely accessible Load or reuse scan settings Upon starting the software the settings of the previous scan will be loaded automatically In order to use settings of earlier scans use the File Open Scan command By default the file selection dialog will Scan Acquisition Software Chapter 2 Short Step by Step Guide show the settings directory of the user currently logged onto the system Navigate to the respective screen directory and open the file experiment_descriptor xml o The experiment descriptor xml of a previous scan will use the filter and objective positions specified t
44. eeeseeenseeeeeseaees 44 5 1 1 Plates with Predefined Starting Position 222220000222020000nnnnnnne een 44 5 1 2 Slides without Predefined Starting Position ssseeuuse 44 5 2 Systems without Hardware Autofocus ccccssscccecessseeeeeceeseeeeeeseeeeeeesaaees 46 5 2 1 Plates with Predefined Starting Position eeeeessseeessssee 46 5 3 Autofocus Adjustment during a Scan esnuunuueesnnnnnnneennnnnnnnonnnnnnnnennnnnnnnennnn 47 5 4 Online Image Browsing cccccsssseceecceesseeecceeseeeecseaseeeecseaseeeeesagseesesseaaes 47 5 5 WOnline AhalySisckcnsensn nee ee een 48 44 Chapter 5 Running up a Scan OLYM PUS For scan HR to perform a proper scan it is required that all Autofocus Acquisition and Plate Manager settings in the Edit Scan window are properly set see Chapter 4 Setting up a Scan After starting the scan by clicking Run there are different scenarios depending on e the availability of a ZDC hardware autofocus e the usage of plates with or without predefined starting position These different scenarios are explained in the following chapters 5 1 Systems with Hardware Autofocus 5 1 1 Plates with Predefined Starting Position This is the simplest scenario a system with ZDC hardware autofocus and a plate type with predefined starting position i e either system standard or calibrated plate types Upon a click on the Scan S
45. efore operating the system Keep this manual in an easily accessible place near the Screening System for future reference Special care has been taken to guarantee correct and accurate information although this is subject to changes due to further development of the Screening System Thus the manufacturer cannot assume scan Acquisition Software Chapter 1 Introduction liability for any possible errors We would appreciate reports of any mistakes as well as suggestions or criticism Please refer to the respective hardware component manuals for regulatory compliances 1 2 Technical Support If you find any information missing in this manual or you need additional support please contact an Olympus specialist directly 1 3 Safety Precautions Before operating the screening system make sure that you have carefully read and understood all hardware component manuals and the safety precautions found in the respective manuals If the optional laboratory workbench is available please pay attention to close all doors of the workbench box before starting a screen If the screening system is delivered with a robot always take care to be well off the robots operational range For safety precautions during operation or teaching of the robot please refer to the Hamilton handbook If a collision of the robot with other equipment occurs always switch off the robot controller and manually readjust the robot to the init position before continuing with a
46. er Image name C3 W00019 P00002 200000 TO0000 TR tif Previous Next Delete associated images Refresh directory listing 1344x1024 0 5 16 bit image 608 0 0 intensity clipping Path to experiment description D Testhexperiment_descriptor xml m Status OK Refresh directory listing Well Position Color Z Time Use these buttons to navigate through the images Generate AVI Set a file to export an avi file for time lapse experiments Path to experiment description Set the path to the experiment descriptor file to load other data that were previously acquired 5 5 Online Analysis After starting the acquisition it is possible to directly start the analysis of the experiment To do so start the analysis software and load the experiment descriptor dat file by executing Scan Open Set up a new analysis or load an existing analysis as described in the scan R Analysis Software handbook After the acquisition and analysis are completed it is possible to revisit the objects that were detected during analysis Therefore go to Repositioning Interactive in the analysis software The Interactive Repositioning window opens Select an object in the scan HR analysis software i e click either on one of the data points or on a detected object in one of the image displays main image panel galleries After selecting an object the stage moves back to the position and shows a live view Thereby the object selected in the
47. here If a different hardware configuration is used for the new scan the settings might have to be adapted accordingly see following chapters Load the Well Plates In case of a scan4R system without autoloader robot or if the robot is not to be used place the plate on the stage A system with autoloader robot allows screening several well plates within a single run Up to four stackers are available on the right of the robot One of the stackers has to be empty at the start of the scan and will serve as swap stacker Note that there is an additional position in front of stacker 1 which is used for fine positioning of the plates Keep this position empty You can remove the stackers from their positions to make loading and transportation easier When loading the stackers take care of the following Make sure that the transportation covers are removed from the stackers Orientate the plates with the first column pointing towards the microscope Make sure that each stacked plate fits accurately on the plate below Stack the plates as well aligned as possible Leave a minimum gap of at least 10 mm from the last plate to the top of the stacker to allow the robot to lift the uppermost plate from the stack 4 N Microscope Autoloader Robot Stackers Specify and label the wells and the sample Click the Scan Edit button or execute the File Edit command to open the Edit Scan window Click the Plate Manager tab the
48. ied objective in the objective list Acquisition config ObjectiveSettings xml 6 Continue at point 2 for the next objective If an additional limit is required because of a frame or plate inserted this can be modified in the Border Thickness Fields of the Plate format see Edit Scan Plate manager Edit Plate Types Chapter 4 5 Plate Type Settings Frame Border x Stroke x Stroke y Stage Calibration Corner x y z r l l Focal Plane seus EE 71N q Z N Frame Border z Representation of the frame and objective When setting up the limits the objective parameters r and dZ are evaluated and stored scan Acquisition Software Chapter 7 Appendix 67 7 4 Remote control The program Remote control exe can be found in the scan R program folder in Aquisition Remote control The program allows to synchronize an external hardware device e g a liquid handling system with the scan R Acquisition 7 4 1 Connection to the server The acquisition software is the server The client connects to the server with the TCP IP protocol on the port defined in System System configuration Remote Only one client can connect to the acquisition software When not in standalone mode and when the Edit Scan window is not opened the server is waiting for connection When a client connects all buttons of the scan4R main interface are deactivated except for the Browse button 7 4 2 Communica
49. ion Software Chapter 7 Appendix Not supported with the given specification are Multiple liquid insertions at a single Well Position with subsequent interrupted time lapse acquisition AND AF after the second insertion step Automated Analysis takeover of time lapse ranges time lapse liquid insertion points have to be remembered and set manually in the analysis software Feedback loops are only possible with considerable client programming as scans have to be stopped and newly prepared settings have to be issued Within a single scan no sequences are possible that are different from the regular sequences In order to change the sequence the scan has to be stopped and restarted 13 OLYMPUS OLYMPUS LIFE SCIENCE EUROPA GMBH Postfach 10 49 08 20034 Hamburg Germany Printed in Germany 2008 12 M 2 1 0 14
50. ioning frame in front of stacker 1 from its original position This stacker is modified to allow special safety procedures for accurate and robust positioning and is different from the other stackers 10 Chapter 2 Short Step by Step Guide OLYM PUS scan Acquisition Software Chapter 3 The Main User Interface 3 Main User Interface This chapter explains the features of the image displays and briefly introduces the different menu points and buttons accessible from the main user interface o MEE clause cT TE 12 3 2 Display Context Ment ana ee a a a autos 14 3 3 Uo c EREEREU 16 3 4 Devices Microscope Control and Live View seesesseesssss 17 3 4 1 Microscope Settings nasain e e e 17 SP V2 MEET ool oe eS en MC POOR 18 Soo dx MEN S A vieille eT EM 19 S Menu ea A A DM I De IM Oa lc LEM DA IUE 19 11 12 Chapter 3 The Main User Interface OLYM PUS 3 1 Front Panel Displays oe soan Acguisilion File ese Ogia Syin Abt EEEE PaES 4 JHI eS 6 Pisis kert p NHESSENENE Lx 1ELPR 01 Sot ur 116 rna Dipta AF be Tie na 00 00 herir aT D Progress EBERRREREHRHERERERARARARREREREREM The main window features one large and three small live image displays by default This allows to separately observe different color channels a multicolor channel overlay RGB Overlay Transmission
51. it button or select File Edit Autofocus Acquisition Plate Manager All modifications in the settings persist until they are changed again When starting a scan the complete settings will be stored in the resulting directory in a file named experiment descriptor xml Note that by default the current user settings will be stored in Defau tSettings xm in the in the scan R installation folder in the directory Acquisition Scan Settings Library current user where current user corresponds to the Windows login name This means that when multiple Windows users start the system for each user scan H will load each user s default settings Another filename or folder to store the user settings can be set via File Save as It is also possible to reload a saved set of settings via File Open and load an experiment descriptor xml file or a click on the Open button and change the settings at will The Edit Scan dialog window groups the settings on three tabs e Autofocus to define and optimize the autofocus procedure e Acquisition to define and visually control the microscope camera and illumination settings to set cycle times for scan repetitions and to setup the acquisition of z stacks e Plate Manager to choose the well plate format select the wells and the positions within wells to be imaged 4 1 Autofocus Settings Test Current system settings Click here to open the Autofocus Settings window It allows you to crudely focus and t
52. lder extends over the insert Otherwise use default values 39 40 Chapter 4 Setting up a Scan OLYM PUS Autoloader settings Hamilton method Select a method for the Hamilton plate loading robot from the shortlist Active only if the robot is activated in the Devices and Ports tab of the System Configuration see Chapter 6 1 1 Devices and Ports Use barcode Click here to use a barcode reader for the identification of the plates 4 5 1 Well Measurement scan HR requires information about the position of the first well A1 of the plate in order to start the scan All other positions are then defined by the values given in the Plate Type Settings Often it is sufficient to determine the position of the well center roughly but in some cases a more precise measurement is necessary The two different procedures are described in the two following chapters Use the measurement result always for this well plate type Check this check box if you do not want to repeat the A1 well measurement see below for each scan The stored position will then be used each time this plate type is selected Once the measurement is run a message in light green will be displayed right of the Type box in the plate type settings window A1 position calibrated Tvpe 48 Well best 41 position calibrated o For convenience it is recommended to select Types with defined first well positions blue green background whenever possible even if no autoloader rob
53. lip GXO aisi iscr hoe pace Id edocet 7 4 Remote Control cccccscccccsseceessseeeeseeseeeneeees 4 1 Connection to the server 7 4 2 Communication interface concept 7 4 3 OOmDadssgusatstusemixtmt ad tante maius Chapter 7 Appendix 7 4 4 Known use cases and corresponding communication samples 70 59 60 Chapter 7 Appendix OLYM PUS 7 1 Data Storage Format All the data for a specific scan is stored in the respective storage directory defined on the Acquisition tab of the Edit scan window The storage scheme is as follows screendg Experiment_descriptor dat Experiment_descriptor xml Data B1 W00007 PO0001 Z00000 TOOOO00 alexa tif B1 W00007 POO001 Z00000 TOO000 dapi tif B1 WO0000 7 POQ0002 ZO00000 TO0000 alexa tif ScreenO1 storage directory Experiment_descriptor dat position file Experiment_descriptor xml scan settings file The system parameters are stored in the xml file the stage positions in the dat file A Data subdirectory contains the actual image files in tif format Individual fields separated by a double dash compose the image file names These fields denote in the order of appearance the well comment as shown in the table of the format manager the well index the position index the Z stack index the time loop index and the channel name The names are assembled systematically as a systema
54. m 672x512 1X 250 505 3 3 Focus the specimen using the Z drive tools 4 Click Select to set the starting Z position for the autofocus 5 Click OK to confirm scan Acquisition Software Chapter 5 Running a Scan 47 5 3 Autofocus Adjustment during a Scan Click the Autofocus Settings button on the main window in order to optimize the software autofocus during a scan The Autofocus Settings window opens The upper box affects only the settings of the object based autofocus The options of coarse and fine autofocus can be tuned and the exposure time of the autofocus channel can be changed Je Autofocus Settings Objects Minimum cell Size M asimum cell Size Mark center F bject Threshold 375 B 80 2 pixels 5D0 4 pixels v Frame o jec C Ignore border objects Coarse autofocus Fine autofocus 100 7 58 20 a0 amp 0 407 204 Hn 0 Current position Coarse focus position 20 40 UL Camera 50 Exposure time ms 100 0 58 20 The controls are explained in detail in Chapter 4 1 Autofocus Settings 5 4 Online Image Browsing Click the Online Observation Images Browse button to browse through stored images of the current scan The Image Browser window opens Use the Previous and Next buttons to navigate through the images Delete associated images Removes associated images e g the images of one color channel 48 Chapter 5 Running up a Scan OLYMPUS HW Image Brows
55. m Set here the distance between the centers of two adjacent well columns and rows respectively Well geometry Enter here the shape circle or rectangle and the diameter of the wells A1 settings A1 position 3 point A1 measurement See Chapter 4 5 1 We Measurement A1 position A1 center position only See Chapter 4 5 1 We Measurement ZDC settings Well bottom Thickness and height Thickness um Set here the thickness of the plate Bottom only necessary for systems with ZDC Well bottom Thickness and height Distance from plate frame bottom um Set here the distance from the bottom corner of the plate to the well bottom only necessary for systems with ZDC Measure Only available when a ZDC for hardware autofocus is installed The values for Thickness and Distance from plate frame bottom can be determined by the ZDC Well bottom Value variations Thickness variation um Set here a value for the thickness variation of the plates used only necessary for systems with ZDC Well bottom Value variations Lower surface variation um Set here a value for the variation of the distance between bottom corner of the plate and the well bottom only necessary for systems with ZDC Material Refractive index Set here the refractive index of the well bottom material only necessary for systems with ZDC Plate border settings Border thickness objective security X Y Border um Enter a value only when the plate or slide ho
56. m limits of movements It is very useful albeit not absolutely necessary to additionally calibrate the stage better said to calibrate the position of the well plates relative to the absolute stage coordinate system This is not a procedure that has to be undertaken routinely it suffices to do it once upon installation of the system Thus the access to this routine is password protected 6 2 1 Well Plate Types There are three different classes of well plate types a System standard well plates These are certain commercial plates with hard coded dimensions using the specifications of the manufacturers Their parameters are not editable in the Plate Manager Edit plate types See Chapter 4 4 Plate Manager Only such plates can be used for the stage calibration Once the stage is calibrated the A1 position of these plates is always determined after stage initialization upon software start and thus does not have to be determined prior to the start of a scan Standard well plates are highlighted blue in the Plate Manager 56 Chapter 6 System Configuration OLYMPUS Plate Type Edit plate types Type 12 Well NUNC 150628 System calibrated format Press new to modify the Format b Calibrated well plates This can be any type of well plate in principle Once such a plate type is calibrated via the Edit Plate Types and the Position A1 measurement the A1 position of such a plate is always determined after stage initialization
57. n OLYM PUS Camera Microscope and Illumination settings See Chapter 3 4 Devices Microscope Control and Live View Exposure time auto set Use the auto set button to obtain a value for the exposure time The auto set button is available only when the Autofocus settings window is opened via the Test button The exposure time is set such that the mean intensity of the objects equals a certain predefined value Note that this value is much less than the value specified for the image acquisition channels see Chapter 3 4 1 Microscope Settings and Chapter 4 2 Acquisition Settings to reduce the time necessary for the autofocus procedure When the original image was saturated it may be necessary to press the several times in order to set the exposure time properly 4 1 1 Coarse Autofocus The following settings are interdependent and the software will adjust the controls using the equation Scan range Layers x Step width Scan range Use one of the sliders or type in a value to set the imaging range for the coarse autofocus Make sure that the range is large enough to encompass all possible variations of focal positions caused by the unevenness of the substrates o If the scan range is set too large it may be possible that the autofocus focuses on the wrong surface for example on dust particles underneath the plate Step width um The step width between neighboring Z positions is set automatically to four times the Calculated depth
58. n Spacing Well geometry A1 position 24 Well NUNC 142475 Column spacing um Well shape 48 Well NUNC 150687 Columns pacing um P 2 Circle v Slide 12 mm 8 A 112920 Slide 15 mm Rows Row spacing um Well diameter uim Slide 25 mm 6 112920 9770 Swa Use the measurement result always For this well plate type e 24 Well 48 Well 96 Well Well bottom Border thickness objective security 384 well 96 Well COSTAR 3599 Z measured Thickness um Thickness variation um X Border um Scan specific 1220 l u3 5 Refractive index 0 1 96 Well COSTAR 3596 test Uncalibrat zs wj i LJ 159 LJ Y 3 point 41 measurement Fi A1 center position only Thickness and height Value variations Material 24 Well Zellkontakt Distance from plate 96 Well Greiner SensoPlate WaterImrr frame bottom um Lower surface variation um Y Border um 48 Well test 464 Use 600 0 Measure Autoloader NONE Hamilton method Use Barcode Barcode file Click Edit plate types in the Edit Scan Plate Manager tab to open the Plate Type Settings window Here new plates can be created and existing plates can be changed Geometry of the plate and A1 position As the settings for the ZDC are depending on the plate type used the ZDC related settings can also be found here Well bottom box When an autoloader robot is used to place the pla
59. n no event shall Olympus Soft Imaging Solutions GmbH be liable for any indirect special incidental or consequential damages arising out of purchase or use of this manual or the information contained therein No part of this document may be reproduced or transmitted in any form or by any means electronic or mechanical for any purpose without the prior permission of Olympus Soft Imaging Solutions GmbH 2006 2008 by Olympus Soft Imaging Solutions GmbH All rights reserved Manual version 2 1 December 2008 LICENSE AGREEMENT Preamble OLYMPUS SOFT IMAGING SOLUTIONS GMBH is manufacturer of the scan R software and drivers interfacing software to Olympus Soft Imaging Solutions manufactured hardware These components are referred to as OSIS scan R SOFTWARE PRODUCT LICENSE AGREEMENT between END USER and OLYMPUS SOFT IMAGING SOLUTIONS regarding the OSIS scan4R SOFTWARE PRODUCT IMPORTANT READ CAREFULLY Below you will find the contractual agreements governing the use of the OSIS scan4R SOFTWARE PRODUCT These conditions apply to you the user and to OLYMPUS SOFT IMAGING SOLUTIONS With any of the following actions you explicitly agree to be bound by the conditions of this contract purchasing the software opening the package breaking of one of the seals or using the software In case you do not agree with any of the conditions of this contract please return all parts of the product including manuals without delay Remove all softwa
60. name for each occupied position in filter cube turret Contrast insert Set a name for each transmission condenser insert Emission filter FFWO Set a filter name for each occupied position in the emission filter wheel 6 1 3 MT20 LED LED controller This option is grayed out and out of function in scan H MT20 available Check this box if a MT20 Illumination System is available 54 Chapter 6 System Configuration OLYMPUS fc System Configuration fr System Configuration Devices and ports Microscope MT20 LED Error reporting Remote Devices and ports Microscope MT20 LED Error reporting Remote Objectives LED controller Filter cubes UPLSAPO 10x v E 1 DAJFi TR APO 20x 340 v 2 CFPIYFP MT20 available LUCPLFLN 20x itasse o 3 empty LUCPLFLN 40x 5 empty UPLFLN 60x 6 empty Switch off burner after acquisition F Excitation filter 1 DAPI 350 50 2 FITC 492 18 3 TxRed 572 23 Emission Filter FF WO 1 Filter 1 2 Filter 2 3 Contrast inserts 1 empty 2 empty 3 empty 4 empty 5 empty 6 empty Switch off burner after acquisition Check this box to cause the arc burner to be switched off automatically after the scan is completed o This is a useful option for weekend or overnight operation because it saves burner lifetime Mind however that the burner needs about 10 min on restart to reach full and stable intensity Also switching the burner on and off frequently reduces the lifetim
61. nding on the accuracy required scan Acquisition Software Chapter 7 Appendix 65 Start the Program Acquisition FactoryTools LimitSetup exe and follow the steps as indicated on the dialog T Factory Limit Setup Utility ATTEN TIO N FACTO RY CALI BRATIO N TOOL Activate limits in Scan R acquisition Inproper use might damage the System Joystick required for setup Please check for Objective Stage collision and Stage Lid collision whenever moving Stage or Objective 1 Check the given frame lenghts Frame Stroke X Stroke X um Stroke Y um 124000 86000 DX Border X um Border Y um Border 7 um 2000 0 900 A Corner aq calibrated in Scan R acquisition Border X 2 Select the objective for which vou want to define XYZ limits HER Distance from frame iurandum M eland Dx um DY wm R um Max DX DY DZ um lt 6553 v empty 0 o Lo 3 Move to a frame corner set XYZ maximum and press Calculate R DZ Fine z er z Step size um Current z position 0 um Calculate R DZ Set Defaults 4 Test if the limits are OK Calculated XYZ Limits Maximum XYZ Hardware Limits Calculate and set Limits xmin um ymin um xmin um vein um 0 0 0 0 xmax um ymax um zmax um xmax um ymax um zmax um o jo J jo 0 0 0 Position X um Y um 2 um 0 0 o 5 If the limits are OK press save Save 6 Go to
62. ndow to display only the Status Bar View Main Window This command minimizes the scan R window to display only the largest of the four displays View Maximized This command causes the entire scan R window to be shown 20 Chapter 3 The Main User Interface OLYMPUS O y View gt Log File This command opens the log file The log file contains all executed steps of the scan R system Deactivate the Go to the end of text checkbox in order to navigate through the log file T LOG FILE 48 516 Well 1 Pos 6 2 move to 528720 2 move to 528800 Z move to 528880 2 move to 528960 2 move to 529040 2 move to 529120 Z move to 529200 Z move to 529280 Z move to 529360 Z move to 529440 2 move to 529520 2 move to 529600 2 move to 529680 2 move to 529760 Z move to 529840 Z move to 529280 57 031 End of acquisition 58 312 Screen stopped by user Go to end of text Operate Device Control This command starts live image acquisition and opens the Device Control window that allows to access a selection of hardware settings directly It can also be executed with the Devices button Operate Start Batch This command opens the Batch Selection window to create a list of experiment descriptor xml files and then start a series of scans with different settings iro Batch Selection Batch List D experiment descripkar xml gt Remove Operate Start This command starts a scan It can also be executed with
63. nnn 44 5 1 1 Plates with Predefined Starting Position 2222000000022000nnnnn nennen 44 5 1 2 Slides without Predefined Starting Position sseeeuuse 44 5 2 Systems without Hardware Autofocus eeeeeeseseeeeeeenennennnee 46 5 2 1 Plates with Predefined Starting Position 2222000000022000nnnn nennen 46 5 3 Autofocus Adjustment during a Scan cccccccssseeseeeeeeeeseeesseeeeeeseeseasseeeeeess 47 5 4 Online Image Browsing cccccsssseeeecceeeeceeceeeeeecceeaeeeeceeaaeeeesesagseeeesseages 47 5 5 YOnline amp AnalySis usa nenne aa 48 OLYMPUS 6 System Configuration uu0000000000nn0nnnnnnnunnnnnunnnnnnnnnnunnnnnunnnnnnnnnnunnnnnenn 51 Ol SYSIEM GCONIAUFALON ES 52 5 151 devices ANG Polls Anne een nee 52 Beles MICIOSCODES isee Uer ad qeRecisdub icu at abusi Ra cU Au uM uS Und QUA one dO did QUA Vid 53 55 3 DI 2 WY c ee ee ee ee 53 6 1 4 Error Reporting Remote uu200000020000nonnnnnnonnnnnnnnnnnnnnnnnnnnnnnnnnnnne nee 54 82 Stage AMON AMON ieee ctetu od ette Cit disce iua eben ue is eid eiae deuten 55 ei AVENSIS TYDES penis TEE ea LED 55 6 2 2 Stage Calibration Procedure uuueesssnnnnnnnnnnneennnnnnnnnnnnnnnnnnnnnnnnnnnnennnennnen 56 8 3 Oblectve Partoocallby dass eiie eet ss ot les ences ot ea 57 F SJADDOeDODCos ier epe ore ree Mu dau eite ttp LU ELI UL M 59 FA Data Storage FOMA esae clap iocos
64. ntract 4 OLYMPUS SOFT IMAGING SOLUTIONS is the legal owner of all copyrights and trademarks of the OSIS scan R SOFTWARE PRODUCT and documentation National and international law protect copyrights and trademarks OLYMPUS SOFT IMAGING SOLUTIONS reserves all rights which are not explicitly expressed in written form 8 5 Warranty 1 OLYMPUS SOFT IMAGING SOLUTIONS guarantees for the period of 12 months after the date of purchase that the software works in all major aspects according to the descriptions in the manuals OLYMPUS SOFT IMAGING SOLUTIONS as the producer of the software provides this warranty It does not replace or restrict other warranties or liabilities provided to the User by local or other sales people or organizations OLYMPUS SOFT IMAGING SOLUTIONS does not guarantee that the software is defect free that the software fulfills the specific requirements of the User or that the OBS scan SOFTWARE PRODUCT works with other software provided by the User 2 OLYMPUS SOFT IMAGING SOLUTIONS further guarantees that the software storage devices floppy disks CD ROMs etc and the manuals are free of material defects Defective storage devices or manuals will be replaced free of charge if they are returned to OLYMPUS SOFT IMAGING SOLUTIONS within 90 days of purchase and accompanied by a proof of purchase 8 6 Liability 1 OLYMPUS SOFT IMAGING SOLUTIONS or their sales organizations cannot be held liable for damages or injuries
65. ny system operation Do not use the system in other ways than described in this manual Do not move yourself into the operational range of the robot while the system is screening Pay attention that no liquid enters the system mechanics and electronics when using well plates with liquid content When changing a sample manually always retract the objective via the ESC button on the right of your microscope Never change the filters of the MT20 without wearing soft gloves 3 4 Chapter 1 Introduction OLYM PUS Scan Acquisition Software Chapter 2 Short Step by Step Guide 5 2 Short Step by Step Guide This chapter gives a step by step guide on how to use the scan R software to perform a scan Detailed explanations of the entire setup and acquisition procedure can be found in Chapter 3 Main User Interface Chapter 2 Short Step by Step Guide OLYMPUS Performing a Scan A Short Step by Step Guide If you use new types of well plates new types of samples new staining or staining protocols or if you alter the optical hardware configuration you will most probably have to setup new scan settings to obtain optimal results In that case this step by step guide will be insufficient for your needs The necessary information on how to setup new scan settings can be found in the following chapters Before the start Be sure to have switched on all system devices before starting a scan Start the scan R acquisition software Start th
66. o control if the autofocus channel settings lead to images suitable for autofocusing o When opening the Autofocus settings window the Autofocus tab remains active Move the Autofocus settings windows aside to access the Autofocus tab and change the settings e g exposure time etc Type Select the method to quantify the image focus from the following options scan Acquisition Software Chapter 4 Setting up a Scan e Gradient The focus is determined by analyzing mean intensities and intensity gradients of image structures e Object based The software first detects the objects in each image and then determines the focus by analyzing their mean intensities and intensity gradients The result is weighted with the number of detected objects The objects have to be defined via Object based Test current system settings see Chapter 4 1 5 Object based Autofocus Note that Image acquisition is skipped if no objects are detected to save disk space e Object based coarse on A1 This is the same method as above with the difference that only on the first position of the first well both a coarse and a fine autofocus scan are carried out On all other positions only the fine autofocus is performed This leads to an increased imaging speed however is only suitable for plane samples ti Edit Scan Autofocus Acquisition Plate Manager Current system sett tet lan See _ Interpolate AF start positions Calculated depth of Field 2812
67. oe tra nce aen toni aa een Saxon ut duae 60 1 2 SBROPMOSSSIOS eraut dedo vua que tative ausa t adbuc uo d rato dui adbat uk dona ua dud ul dd RA 60 7 Facto TOOS Rena REEL odd atat duet E E oS ad 63 7 2 EIOBICKUD EX yen re tie eer sea ee een 63 7 3 2 UIT LED x C ernennen eines 64 724 Reno COMM Ol m 67 7 4 1 Connection to the server uueessssnessnnnnneennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnennnnen 67 7 4 2 Communication interface concept u44444444HHH nenn nnnnnnnnnnnnnnnnnnnnn nennen 67 TAS Commands debeat cis dade Cel OM ZI xA CE S c Ed AR AD uo xke d x Ed Sc ed 68 7 4 4 Known use cases and corresponding communication samples 70 scan Acquisition Software Chapter 1 Introduction 1 1 Introduction Thank you very much for purchasing Olympus Soft Imaging Solutions High Content Screening System and for your confidence in our products and service Chapter 1 Introduction OLYM PUS The scan Acquisition Software controls the hardware components of the Olympus scan R Screening Station for Life Sciences The scan Acquisition Software is designed for automated image acquisition of biomedical samples The acquired images can be optimally analyzed with the scan Analysis Software The software is intended for the use in biomedical research IN The scan Analysis Software the scan Acquisition Software as well as the hardware components of the Olympus s
68. of field see next Chapter 4 1 2 F ne Autofocus However it can be changed at will here Layers This is the number of layers to be imaged during the coarse autofocus scan It is automatically calculated from Step width and Scan range Interpolate AF start positions See Chapter 4 1 4 Autofocus with Interpolated Starting Positions Step by step In order to avoid having to run a coarse autofocus scan at each well it is possible to interpolate a plane of focal positions over the entire well plate The corresponding Z value at each well will then be used to start the fine autofocus scan The plane is fitted to the values found using the Collect reference positions button Collect reference positions See Chapter 4 1 4 Reference positions for the plate can be collected in order to either interpolate AF starting positions for all wells or to measure maximum and minimum focus position for selected positions in order to determine a value for the coarse autofocus range scan Acquisition Software Chapter 4 Setting up a Scan 4 1 2 Fine Autofocus Calculated depth of field This is the theoretical depth of field as determined by the NA of the selected objective It does not make sense to set the step width significantly smaller than this value A rather crude approximation is used for the calculation 450 NA nm This approximation is theoretically reasonable only for low NA values and a wavelength of 500 nm However it leads to useful step
69. ot is used This avoids having to set the scan starting position before each scan 4 5 1 1 Well measurement A1 center position only A1 center position only Choose this option if for this experiment and plate type it is sufficient to roughly set just the center position of the first well e g if only central positions of each well are to be measured Measure Click here to open the Plate Measurement for manual plate loading window and proceed as follows YE A1 position for manual plate loading A e e e eo 1 Click Live view Live view 2 Move to well A and center it 3 Confirm as scan starting position with OK eo0e0 veo 1 Click Live view to open a live image window scan Acquisition Software Chapter 4 Setting up a Scan 41 2 Move the center of the first well into position 3 Confirm by clicking OK The Plate Measurement for autoloader robot window will open Click Cancel if no autoloader robot is used The second measurement is not required in this case An autoloader robot loads the plates by turning them by 180 This has to be accounted for in the measurement Proceed as follows if an autoloader robot is to be used TE A1 position for autoloader robot Live view e e e e j 1 Mount the plate turned by 180 2 Click Live view 3 Move to well and center it e eo e e g 4 Confirm as scan starting position with OK Click Cancel if no autoloader is used 7 eeee 1 Mount the plate t
70. pixels 475 pixels C Ignore border objects Detected 68 Set current ROI Set current ROI OK Cancel It is possible to use regions of interest ROIs to set the maximum and minimum size To do so click the ROI button rectangle icon next to the bottom right corner of the live image and draw a green rectangle to define the respective ROI Click Set current ROI to adopt the values of the ROI Minimum cell size Set current ROI Click here if you have defined a ROI that determines the minimal size of the objects Maximum cell size Set current ROI Click here if you have defined a ROI that determines the maximal size of the objects Mark center Activate this option in order to mark the center of each detected object with a red dot Frame object Activate this option in order to mark each detected object with a red bounding frame Ignore border objects Activate this option in order to have objects touching the image borders ignored 1 Select the Object based autofocus procedure 2 Select suitable Camera Microscope and lllumination settings 3 Click the Test button on the Edit Scan Autofocus tab to open the Autofocus Settings window 4 Focus manually using the Z focus wheel on the microscope or use the Z focus tools see Chapter 3 4 2 Z Focus in the software to focus the sample You can use Ocular View to switch the microscope from camera view to ocular view When using the Z focus wheel on the microscope to set th
71. point 2 for another objective 1 Check the given frame lengths When the dialog appears the first time it will show the defaults values for Stroke and Border Check if these defaults are the correct ones when you compare them with the construction drawings of the used frame in Scan H If you have more precise values in your drawing correct these values 2 Select the objective for which you want to define the XYZ limits Start with one objective for which you want to define XYZ limits To do that select the revolver position where the objective is instaled and select the corresponding objective from the list You may physically unscrew the objective and exchange it by another one if required This step will define the objective for which the limits are set 3 Move to a frame corner This is the adjustment step for the objective You can modify Z with the cursors and XY with the joystick to adjust the limit position for the objective For control reasons the distances DX DY are also displayed and for safety MAX DX DY is taken as Objective Radius for the limit setup 66 Chapter 7 Appendix OLYMPUS 4 Test This allows checking if the limits are OK To do this press the button Test Limits The hardware limits will then be applied You can now carefully move with the joystick to check if the objective stops at the required positions 5 Click Save if the limits are OK This will store the frame settings and the values R dZ for the specif
72. position AST AUDOIOCUS SELINGS Arena 22 Add Coarse AULOIOCUS seinen 24 4 1 2 Je ABOIOCLSacdsn iacu donem denote aUi adus E E ERR 25 4 1 3 Gradient Autofocus Adjustment Step by Step sss 25 4 1 4 Autofocus with Interpolated Starting Positions Step by step 27 4 1 5 QObJject based AULOTOGUS oidiecanid sete deaur Eod eed ua ea d eta aaa 28 42 JAGQuISIOF Settings used oum cde cca ioc asa ca eaa eure 30 4 3 GING Olli Eure di atu docto ee ee ei 33 4 4 Plate Managers Sesam ee ee ee 35 4AT Wellrpaten EE UT 35 4427 PosSItIOnSs IN i e 36 4 5 Plate Type Sellingsau n u a n aaa 38 4 5 1 Well M aSUreMeh ccccccecececscscececececececuceauacacscscecececececeauauavavaeseseeeecenenes 40 21 22 Chapter 4 Setting up a Scan OLYM PUS The scan4R Screening Station is a complex system with a number of hardware devices and a wide range of experimental possibilities In order to perform a scanning experiment a complete set of parameters is required with information that allows the controller to synchronize the motorized microscope modules the illumination source and the motorized stage with the camera that configures the autofocusing procedure and last not least defines the type of imaging experiment to be executed at a precise pattern of positions on the plates and within the wells The Edit Scan dialog window allows you to modify and verify all settings To open it click the Ed
73. r 4 Setting up a Scan Time lapse settings Loop Select the kind of time lapse experiment for the whole plate for a well or on each single position of a well Time lapse settings Cycles Set the number of times the entire plate scan is to be repeated Time lapse settings Cycle time Set the repetition time If this time is smaller than the time required for a single run the repetition will be performed as fast as possible without delay Image acquisition Binning The CCD chip is composed of many light sensitive units pixels These pixels can be read out individually binning 1x1 or the signal of neighboring pixels can be combined electronically during data readout binning 1x1 Binning reduces the spatial resolution but increases the sensitivity and thus reduces the exposure time required for a good signal to noise ratio It further reduces the amount of data and consequently increases the readout speed Therefore binning is recommended if weak signals have to be detected at high acquisition rates and if spatial resolution is of minor importance Storage Directory Set here the general scan data directory The data for each individual plate will be stored here in a subdirectory with the same name as the plate 4 3 Live Settings Click the Live settings button in the Edit Scan Acquisition tab to come to the Live settings window that allows you to observe the changes of the settings on the image acquired by the camera 1 If
74. re installations of the product from any computer you might have installed it on Return all electronic media of the product or completely destroy all electronic media of the product and send proof that this has been accomplished For a refund please return everything to where you purchased the product 1 Scope 1 This License agreement explicitly covers only the software diskettes or other media you received with the purchase and the software stored on these media the manuals as far as they were developed and produced by OLYMPUS SOFT IMAGING SOLUTIONS 8 2 User rights 1 OLYMPUS SOFT IMAGING SOLUTIONS permits the User for the duration of this contract to use the software on a single computer and a single terminal on that computer This license is explicitly non exclusive i e the User does not have an exclusive right to use the software As a licensed user you can copy the software from one computer to another by using a computer network or other storage devices as long as it is assured that the software can only be used on a single computer or terminal at any time and that the conditions set forth under S 4 are observed 2 The User has the right to produce a copy of the software only for backup purposes 8 3 Additional user rights Only if OLYMPUS SOFT IMAGING SOLUTIONS provides the User with permission in written form the User can incorporate parts of the software into other software developed by the User A distribution
75. resulting from the use of the software or the lack of capabilities of the software unless the User can show gross negligence on the part of OLYMPUS SOFT IMAGING SOLUTIONS This applies without exceptions also to losses of productivity or profit interruptions in the flow of business or manufacture loss of information and other financial losses Without exceptions the possible liability of OLYMPUS SOFT IMAGING SOLUTIONS is limited to the amount that the User paid for the product These limitations on the liability do not influence claims for reasons of product liability 8 7 Contract duration legal consequences of violating the license 1 The contract is deemed to be in force for an unspecified period The User rights are automatically terminated if one of the conditions of the contracts has been violated 2 In case of a contract violation the User has to return the original storage devices and all copies thereof including all modified copies and all printed and written documentation to OLYMPUS SOFT IMAGING SOLUTIONS or the User has to destroy these items 3 In addition OLYMPUS SOFT IMAGING SOLUTIONS reserves the right to file a lawsuit to claim reparations for damages non compliance or removal of the software in case of license violations The following laws and or conditions are in effect the conditions of this contract copyright laws and the laws of the civil code scan Acquisition Software Contents Contents 1 INIFOQUC
76. rol window opens fr Edit Scan Autofocus Acquisition Plate Manager Selected Color Channels Live settings DAPI A fitc TexasRed Name DAPI Microscope Camera Illumination Objective Exposure time ms Fluorescence UPLSAPO 10x 99 Y Transmission Filter cube Preacquisition delay s Excitation filter Emission filter DAJFi TR 0 s DAPI 350 50 v Filter 1 New Remove Defined Color Channels DAPI v 0 00 Z offset um Light intensity 100 1 Add to list Delete Z stack settings Time lapse settings Image acquisition Lavers Loop Binning 1 Projections Plate v 1x1 v Step width km Cycles Cycle time h min s 0 1 lt 00 00 00 Storage directory D Test im Microscope Objective Select the objective to be used Note that a switch of objectives during an experiment is possible but rather time consuming In order to maintain the same focus position for different objectives use the Objective Parfocality setup see Chapter 6 3 Objective Parfocality Microscope Filter cube Select the filter cube to be used Note that a filter cube switch is time consuming even if the system features the fast IX2 FRFACA motorized filter cube turret Imaging speed can be increased considerably by using multi band filter sets Microscope Z offset um This allows setting a Z position offset for the image acquisition relative to the position found by the autofocus The offset is useful for imaging str
77. s start value for the coarse autofocus and performs coarse and fine autofocus according to the ranges set in the Edit Scan Autofocus tab 8 The values given as Z min Z max Z range can also be used to adapt the range of the coarse Autofocus This way the range of the coarse autofocus can be set to match the difference between the maximum and the minimum value of the reference positions In order to minimize the possibility of wells to be outside of the scan range an extra margin to the scan range of the coarse autofocus can be added 4 1 5 Object based Autofocus The Object based Autofocus algorithm first detects the objects in each image and then determines the focus by analyzing their mean intensities and intensity gradients The weighting function of the coarse autofocus is calculated from the sum of all objects sharpness functions and is very sensitive to the number of objects detected The Object based Autofocus properties must be adjusted carefully to ensure that the number of objects is maximal when the image is focused The Object based autofocus may be suitable if a certain structure placed in a certain Z layer within the sample is to be focused e g small cells growing upon larger cells By setting min max cell size accordingly it can be ensured that the desired objects are used for autofocus o Note that the Object based Autofocus properties are important also because images void of detected objects will not be stored on
78. scan H analysis software is centered Scan Acquisition Software Chapter 5 Running a Scan 49 De Interactive Repositioning Oe Interactive Repositioning Oscar lees oiis 1 00 Select the color channel to be displayed in from the short list The sample can be focused with the Z control in case a Z drift occurred in between the first measurement and the repositioning The Ocular view allows to inspect the hits detected by the analysis by eye In the case that the scan R analysis software is running on a different computer than the acquisition software it is necessary to set the IP address Allowed hosts and the port use default 2055 of the computer where the analysis is running see Chapter 6 1 4 Error Heporting Hemote In the scan R analysis software open Settings and in the Repositioning amp Reclassification box enter the Port of the Acquisition Server 2055 as well as its IP Address see also scan R analysis Instructions Chapter 2 3 General Settings 50 Chapter 5 Running up a Scan OLYM PUS scan Acquisition Software 6 System Configuration This chapter explains how the system devices have to be configured in order for scan4R to work properly 6 1 System Configuration 6 1 1 Devices and Ports 61 2 MICR SCOBE een eko MT20J LED esse 6 1 4 Error Reporting Remote 6 2 Stage Calibration
79. ss to system initialization again HW AF ZDC activated but HW AW or IX2 not found Please deactivate in Settings Factory Setup File Not found Leaving Program No configuration file found Please configure your system Start of screen Error Message Robot instruction found but robot not configured properly Please select a proper destination directory in your settings Scan aborted An error occurred during robot operation Please open the case and move the robot to the initialization position Please remove your plates from the microscope and empty the stack Scan aborted If the robot is configured not more than one plate starting position is possible You have not taught any position No Scan will be started Chapter 7 Appendix Data bits 8 Parity N Stop bits 1 Flow control NONE For detailed instructions on how to set the appropriate parameters please refer to the hardware manual Check that you have connected the microscope controller UCB to the serial port of your computer as configured in Settings System configuration Make sure that the UCB is turned on Check if the PC serial ports are set correctly in Settings System configuration scan R uses the following values Bits second 19200 Data bits 8 Parity E Stop bits 2 Flow control NONE For detailed instructions on how to set the appropriate parameters please refer to the hardware manual Move the objective manuall
80. t button Hesponses StartExperiment DONE StartExperiment DENY Screen is running StartExperiment ERROR cancelled by user wrong settings for example not enough hard disc space When not enough hard disc space left screen is aborted StopExperiment It stops the experiment like the stop button Hesponses StopExperiment DONE StopExperiment DENY No screen is running scan Acquisition Software Chapter 7 Appendix 69 SetExperimentCluster filename Changes the current settings like the open button when no screen is running The experiment comes from a filename The filename is relative to the directory Scan Settings Library Examples CLIENT SetExperimentCluster test xml SERVER SetExperimentCluster test xml DONE CLIENT SetExperimentCluster Administrator test xml SERVER SetExperimentCluster Administrator test xml DONE CLIENT SetExperimentCluster test not existing xml SERVER SetExperimentCluster test not existing xml ERROR File not found CLIENT SetExperimentCluster test xml SERVER SetExperimentCluster test xml DENY Screen is running GetNumber Argument Argument is one of the following Wells Subpositions Timeframes Examples CLIENT GetNumber Subpositions SERVER GetNumber Subpositions DONE 4 CLIENT GetNumber Timeloops SERVER GetNumber Timeloops DENY Argument not known RunTo Argument Argument might be one of the following ExperimentEnd PlateEnd Well XY Well XYZ Subposition XY Subposition XYZ
81. tart button scan H conducts the entire scan automatically as set up in Edit Scan and stores the focused images in the predefined folder 5 1 2 Slides without Predefined Starting Position In case of commercial plates of one and the same type one can in most cases assume that upon mounting on the stage the position of well A1 will always be at the same position and the following scenario will not apply In case of spotted slides and non calibrated plates however scan R needs to be taught the starting position each time anew 1 Click Scan Start to start the scanning routine The following message will appear Ti A1 position 1 2 3 4 A e e e e Well A1 position has not been set o cO o Use previous Cancel Use previous This option is only useful if a scan is to be repeated with the same slide immediately scan Acquisition Software Chapter 5 Running a Scan 45 Ti Scan Starting Position No starting position For the scan has been set 1 Move to the center of the first upper left well 4 Repeat these steps For each slide if more than one plate is mounted 2 Focus the specimen after setting the next slide position 3 Click Select to set the starting position 5 Click OK to confirm P zfocus XY navigation Mi Coarse Fine Step size 3 00 um Current position m 5259 57 um 20 sin 096 clipping Clipping 0 v starting position For slide 1 Ocular view 672x512 1
82. tes onto the microscope the robot method can be selected in this window as well Autoloader box The Autoloader parameters define the automation method for an autoloader robot The functions are active scan Acquisition Software Chapter 4 Setting up a Scan only if the robot is activated in the Devices and Ports tab of the System Configuration see Chapter 6 1 1 Devices and Ports On the left side of the window the currently available plates are listed New plate type definitions that are not available in the plate type list can be imported with System Import Plate Types The light blue marked plates are calibrated plates The settings of the calibrated plates can not be changed The message System calibrated format Press new to modify the format is shown right to the plate type The settings for light green and white plates can be changed For light green plates the A1 position has already been calibrated see Chapter 4 5 1 Well Measurement whereas for plates that are not calibrated white the A1 position has to be set when the scan is started Delete Click here to remove the selected custom plate type from the shortlist Standard plate types cannot be removed New Creates a new plate type Type Enter here the name of the well plate type as it is to be listed in the shortlist Plate geometry settings Columns Rows Set here the number of columns and rows of wells on the plate respectively Column Row Spacing u
83. the Coarse autofocus Scan range in the Edit Scan Autofocus tab or try to find a more reasonable starting position 4 4 Plate Manager The Edit Scan Plate Manager tab provides the functions needed to define the pattern of wells and the pattern of positions within each well to be imaged during the scan 4 4 1 Well pattern The circles or squares in the scheme represent the wells of the selected well plate type Blue circles are wells activated for scanning gray circles are wells to be skipped Plate Enter a name in the box If the plate name is unchanged in the course of consecutive scans the scan directories will be stored with an incremented numeration appended to the given name Type Select the well plate format from the shortlist of standard plate types It is possible to add custom plate types via Edit plate types and the Plate Type Settings window see Chapter 4 5 Plate Type Settings Edit selection table This opens the Well Description window with the list of selected wells You can enter a name or description for the selected wells It is also possible to Import a well selection 36 Chapter 4 Setting up a Scan OLYMPUS tk Edit Scan HN Autofocus Acquisition Plate Manager Well pattern Positions in well Del key removes selected positions Fill Pattern Spacing Columns Column spacing um 2 g 867 5 Rows Row spacing um 660 g skip Meander wells Well diameter um 12100 Left
84. the Start button System gt Initialize Devices This command performs a hardware component check It is necessary for example if a device is switched on after the software has been started System gt System Configuration This command opens the window that allows configuring the hardware modules See Chapter 6 System Configuration System Stage Calibration This command starts the stage calibration See Chapter 6 3 Stage Calibration System Import Plate Types This command allows importing new plate type definitions that are not available in the plate type list System gt Objective Parfocality This command allows you to set an offset for the Z position for the used objectives so that when changing the objective the focus is maintained Therefore enter a value for Parfocality um to correct the focus shift for the objectives relative to the objective which you used for the autofocus cf Chapter 6 3 Objective Parfocality About This command gives system and software information scan Acquisition Software Chapter 4 Setting up a Scan 4 Setting up a Scan This chapter gives a detailed description on how to customize scan settings and how to setup the system for maximum performance and reliability The necessary settings encompass the selection of appropriate microscope illumination and camera settings the Autofocus setup the parameterization of the well plates and the details of the imaging to be performed at each
85. tic train of parameters separated by double dashes in the following order well name Comment well index position index Z layer index time loop index and the channel name 7 2 Error Messages Devices initialization Error Message Explanation Possible Measures No connection to controller Retry timeout There is a communication problem with the real time controller Make 70s Cancel sure that it is installed and configured properly with ObsConfig exe and ObsUpdate exe located in scan R acquisition directory And then Connection to real time controller RTC failed Camera not responding Please check your There is a communication problem with the CCD camera Make sure installation and cable connections that it is installed properly and appears in the hardware control panel of the computer Check your cable connections Stage not responding Please check your Check if the stage controller is connected to the serial port of the PC cable connections and your serial settings as configured in Settings System configuration Make sure that the controller is turned on Check if the PC serial ports are set correctly in Settings System configuration scan R uses the following values Bits second 19200 scan Acquisition Software UCB not responding Please check your cable connection and your serial settings UCB Could not move objective to lower position before stage calibration Please reset the UCB and proce
86. tion interface concept Syntax Client Request Command name arguments carriage return linefeed This has to be sent within 6 seconds Server Answer Command name arguments OK or ERROR or DENY response or error description carriage return linefeed DONE The command was successfully executed DENY The command cannot be executed the syntax is not correct or the time point is not correct When a previous command is being executed it continues to run When no command was being executed it waits for the next command ERROR An error occurred while executing the command Error descriptions Command not known Argument not known 68 Chapter 7 Appendix OLYM PUS Argument over range Screen is running N screen is running Waiting for end of previous command RunTo Argument not reached until end of experiment File not found Empty message others errors are written in the logfile Screen continues Screen ended During a screen when an error occurred the error descriptions screen continues or screen ended indicate if the screen is aborted or not The communication is logged in the viewable logfile with following preceding Server Client TAG SERVER CLIENT 7 4 3 Commands Quit The connection is closed by the server GetComlnterfaceVersion Returns the interface version number Hesponse GetComlnterfaceVersion DONE 1 StartExperiment Starts the experiment like the star
87. uctures that are located in a different Z position within the sample than the position found by the autofocus The Z offset is most conveniently set interactively in the Live Settings window see Chapter 4 3 Live Settings Camera Exposure time ms The exposure time determines the period of time during which the CCD chip is sensitive to incoming light in other words during which photons are collected and converted into charges to be read out afterwards Enter a value manually or use the Auto set button Auto set Use the auto set button to obtain a value for the exposure time The auto set button is available only when the Live settings control window is opened via the Live Settings button The exposure time is set such that the mean intensity of the objects equals a certain value When the original image was saturated it may be necessary to press the several times in order to set the exposure time properly Note that when using the Auto set button in order to set the exposure time for 32 Chapter 4 Setting up a Scan OLYMPUS different assays the value set for the intensity threshold object detection algorithm of the scan R Analysis software will be the same for these assays Camera Preacquisition delay ms You may set a delay to be applied before the color channel is acquired Enter a preacquisition delay e g if transmission light intensity is to be changed during acquisition to wait for the light intensity to reach a constant
88. urned by 180 as depicted in the scheme 2 Click Live view to open a live image window 3 Move the center of the first well into position 4 Confirm by clicking OK 4 5 1 2 Well measurement 2 3 point A1 measurement 2 3 point A1 measurement Choose this option if the experiment and the plate type require a precise determination of the first well position e g when using full in to measure all positions of one well Depending on the well shape rectangular circular two or three points have to be set In case of a rectangular well shape the two opposite corners of the well have to be set In case of a circular well three points on the border of the well have to be determined Measure in case of 2 3 point A1 measurement Click here to open the Measure Well window and proceed as follows In case of circular wells 1 Move the border of the first well to the red cross center in the live image window 2 Click Add Position 3 Repeat this for one two more well border positions 4 After the second third position has been added the numbers field next to the Add Position button turns red to indicate that the measurement is completed Confirm by clicking OK 42 Chapter 4 Setting up a Scan OLYMPUS p J haint A1 measures I More Ehe sage in Eros vendi AT bordar H pe hen rom croce T Ck Fucked Pot 3 Riga this l or bres other Ab border posa 4 C rfin wth Ok Zeus XY rurigation e Expriionn enn ii 7
89. width values for objectives with high NA as well Set as step width Click here to set the Calculated depth of field as step width between neighboring Z positions of the fine autofocus scan The number of Layers will be adjusted automatically o This function additionally sets the step width of the Coarse autofocus to four times the Calculated depth of field and adjusts the number of Layers accordingly The following settings are interdependent and the software will adjust the controls using the equation Scan range Layers x Step width Scan range Use one of the sliders or type in a number to set the imaging range for the fine autofocus Reasonable values to start are 10 um and 10 um Step width um You may set here a value different from the Calculated depth of field as step width to be made between neighboring Z positions during the fine autofocus scan Layers This is the number of layers to be imaged during the fine autofocus scan It is automatically calculated from Scan range and Step width 4 1 3 Gradient Autofocus Adjustment Step by step The Gradient Autofocus algorithm determines the focus by analyzing mean intensities and intensity gradients of image structures 1 Select the Gradient autofocus procedure 2 Select suitable Camera Microscope and Illumination settings 3 Click the Fine autofocus Set button to automatically set the Fine autofocus Layer Step width 4 Set the Fine autofocus Scan range to 10
90. x228 549 1 Eram 2 Click Set to open the Scan Starting Position live image window 3 Move to the center of the first upper left well 4 Focus the specimen using the Z drive tools 5 Click Select to set the starting position for slide 1 6 Repeat these steps for each plate if more than one plate is mounted Make sure to set the correct slide number each time 7 Click OK to confirm 8 scan H conducts the entire scan automatically as set up in Edit Scan and stores the focused images in the predefined folder o Note that the positions you define refer to the center of well A1 of the well pattern and thus not necessarily to the first well activated for acquisition in the Edit Scan Well Manager window 46 Chapter 5 Running up a Scan OLYM PUS 5 2 Systems without Hardware Autofocus 5 2 1 Plates with Predefined Starting Position 1 Click Scan Start to start the scanning routine The following message will appear You have bo set an initial z focus position Use previous Cancel Use previous This option is only useful if a scan is to be repeated with the same plate immediately 2 Click Teach new to open the Autofocus Starting Position live image window ic Autofocus Starting Position The software autofocus requires a starting position 1 Focus the specimen 2 Click OK to confirm Fine Step size 3 00 um EZ Current position 5222 03 um 0 2000 4517 Clipping l Histogra
91. y from the lowest position Reset the UCB and process to system initialization again No Hardware autofocus was found on the system After the devices initialization deactivate the checkbox ZDC present in System system configuration The config file FactorySettings xml was not found The software cannot start Proceed to installation of the Scan4R setup No default settings for a scan were found Configure or Proceed to installation of the Scan H setup e A position file with autoloader robot instructions is found but the robot operation has not been configured in the New Edit Scan window of Edit Scan Format Format Manager your configuration Enable robot operation in Settings System configuration You selected a custom position file in your Plate Manager settings This file does not exist The selected storage directory does not exist You have not selected any destination directory The movement of the robot was interrupted This occurs when there is an obstacle in the movement path of the robot Make sure that the robot can move freely Turn off the robot controller and move the robot to the initial position An error occurred during the initial steps of a scan The robot detected a plate on the stage or on any stack assumed empty Remove the plate You tried to configure more than one plate on a single sample holder combined with automatic robot loading The selection of more than one plate on a single

ScanR acquisition

Contents

Download Pdf Manuals

Related Search

Related Contents