Инструкция: NA OLYMPUS SCAN R ACQUISITION

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INSTRUCTIONS

AUTOMATED IMAGE ACQUISITION

SOFTWARE

This instruction manual describes Olympus

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

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.

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® INSTRUCTIONS AUTOMATED IMAGE ACQUISITION SOFTWARE This instruction manual describes Olympus 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
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OLYMPUS SOFT IMAGING SOLUTIONS GMBH Rupert-Mayer-Strasse 44 D-81379 München Tel: +49 89 - 89 55 805 660 Fax +49 89 - 89 55 805 6606 Email: info@olympus-sis.com www.olympus-sis.com
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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
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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
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Acquisition Software Contents Contents 1 1.1 1.2 1.3 2 Introduction ...............................................................................................1 Abstract ..........................................................................................................2 Technical Support
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6 System Configuration.................................................................................51 6.1 System Configuration .................................................................................. 52 6.1.1 Devices and
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Acquisition Software Chapter 1 –Introduction 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. 1
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2 Chapter 1 – Introduction The Acquisition Software controls the hardware components of the Olympus scan^R Screening Station for Life Sciences. The Acquisition Software is designed for automated image acquisition of biomedical samples. The acquired images can be optimally analyzed with the Analysis
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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
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4 Chapter 1 – Introduction
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Acquisition Software Chapter 2 – Short Step-by-Step Guide 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. 5
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6 Chapter 2 – Short Step-by-Step Guide 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
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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. The experiment_descriptor.xml of a previous scan will use the filter and
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8 Chapter 2 – Short Step-by-Step Guide 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
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Acquisition Software Chapter 2 – Short Step-by-Step Guide • If no ZDC hardware autofocus is available, scan^R requires the user to set interactively a starting Zposition for the software autofocus. Afterwards the scan will be carried out automatically. • If spotted slides or other sample holders
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10 Chapter 2 – Short Step-by-Step Guide
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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. 3.1 3.2 3.3 3.4 3.4.1 3.4.2 3.4.3 3.5 Front-Panel
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12 Chapter 3 – The Main User Interface 3.1 Front-Panel Displays 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) or an online view of the
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Acquisition Software Chapter 3 – The Main User Interface 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
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14 Chapter 3 – The Main User Interface 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
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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 selected color palette. Percentage values can be set between 0% and 5%. 1% is usually
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16 Chapter 3 – The Main User Interface 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
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Acquisition Software Chapter 3 – The Main User Interface 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
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18 Chapter 3 – The Main User Interface Microscope: Filter cube. Select the filter cube to be used. Note that a filter cube switch is timeconsuming 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
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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 size. Z focus:
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20 Chapter 3 – The Main User Interface View 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. Operate Device Control. This command starts live image
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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,
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22 Chapter 4 – Setting up a Scan The scan^R 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
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Acquisition Software Chapter 4 – Setting up a Scan • Gradient. The focus is determined by analyzing mean intensities and intensity gradients of image structures. • Object-based. The software first detects the objects in each image and then determines the focus by analyzing their mean intensities
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24 Chapter 4 – Setting up a Scan 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
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Acquisition Software 4.1.2 Chapter 4 – Setting up a Scan 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
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26 Chapter 4 – Setting up a Scan 7. Once finished, click the Test button. 8. The Autofocus settings live image window will open and display a live image acquired with the settings of the Autofocus tab... 9. Focus manually using the Z-focus wheel on the microscope or use the Z-focus tools (see
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Acquisition Software 4.1.4 Chapter 4 – Setting up a Scan 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
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28 Chapter 4 – Setting up a Scan 5. Repeat the Z-focus measurement at two or more different stage positions that are reasonably far apart. 6. From these measurements scan^R will interpolate a grid of starting positions. 7. At each measurement position the software autofocus takes the interpolated
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Acquisition Software Chapter 4 – Setting up a Scan 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
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30 Chapter 4 – Setting up a Scan 5. 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
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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
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32 Chapter 4 – Setting up a Scan 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.
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Acquisition Software Chapter 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
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34 Chapter 4 – Setting up a Scan 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
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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
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36 Chapter 4 – Setting up a Scan . 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.
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Acquisition Software Chapter 4 – Setting up a Scan 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
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38 Chapter 4 – Setting up a Scan 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
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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
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40 Chapter 4 – Setting up a Scan 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
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Acquisition Software Chapter 4 – Setting up a Scan 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
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42 Chapter 4 – Setting up a Scan 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
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Acquisition Software Chapter 5 – Running a Scan 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 5.1.1 5.1.2 5.2 5.2.1 5.3 5.4 5.5 Systems with Hardware Autofocus
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44 Chapter 5 – Running up a Scan For scan^R 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
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Acquisition Software Chapter 5 – Running a Scan 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
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46 Chapter 5 – Running up a Scan 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: Use previous. This option is only useful if a scan is to be repeated with the same plate
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Acquisition Software Chapter 5 – Running a Scan 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
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48 Chapter 5 – Running up a Scan 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
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Acquisition Software Chapter 5 – Running a Scan Select the color channel to be displayed in from the short list. The sample can be focused with the Zcontrol in case a Z-drift occurred in between the first measurement and the repositioning. The Ocular view allows to inspect the ‘hits’ detected by
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50 Chapter 5 – Running up a Scan
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Acquisition Software Chapter 6 – System Configuration 6 System Configuration This chapter explains how the system devices have to be configured in order for scan^R to work properly. 6.1 System Configuration...................................................................................52 6.1.1
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52 Chapter 6 – System Configuration 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
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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
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54 Chapter 6 – System Configuration Switch off burner after acquisition. Check this box to cause the arc burner to be switched off automatically after the scan is completed. This is a useful option for weekend or overnight operation because it saves burner lifetime. Mind, however, that the burner
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Acquisition Software Chapter 6 – System Configuration 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 scan^R Acquisition software is started the stage initializes automatically to determine its maximum limits
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56 Chapter 6 – System Configuration 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 upon software
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Acquisition Software 2. Execute System Chapter 6 – System Configuration 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
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58 Chapter 6 – System Configuration 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.
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Acquisition Software Chapter 7 – Appendix 7 Appendix The following chapter provides additional technical information. 7.1 7.2 7.3 7.3.1 7.3.2 7.4 7.4.1 7.4.2 7.4.3 7.4.4 Data Storage Format....................................................................................60 Error
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60 Chapter 7 – Appendix 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: Screen01: storage directory; Experiment_descriptor.dat: position file;
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Acquisition Software Chapter 7 – Appendix Data bits: 8, Parity: N, Stop bits: 1, Flow control: NONE • 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
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62 Chapter 7 – Appendix • A scan is configured with an autoloader, but the stage pick-up position for the robot was not yet defined • Define the pick-up position with the utility LidPickup.exe Not enough space left to fulfill your request. For robot loading a number of 20 plates is assumed. The
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Acquisition Software Chapter 7 – Appendix • Stage not responding ... • 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.
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64 Chapter 7 – Appendix 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 Scan^R
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Acquisition Software Chapter 7 – Appendix Start the Program: .\Acquisition\FactoryTools\LimitSetup.exe and follow the steps as indicated on the dialog. 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
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66 Chapter 7 – Appendix 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.
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Acquisition Software Chapter 7 – Appendix 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
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68 Chapter 7 – Appendix – Argument over range – Screen is running – No 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
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Acquisition Software Chapter 7 – Appendix 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
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70 Chapter 7 – Appendix 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
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Acquisition Software Server Chapter 7 – Appendix Client GetNumber Wells GetNumber Wells DONE 3 StartExperiment StartExperiment DONE RunTo Well_XY RunTo Well_XY DONE “The automate provides the liquid” RunTo Well_XY RunTo Well_XY DONE “The automate provides the liquid” RunTo Well_XY RunTo Well_XY
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72 Chapter 7 – Appendix RunTo Well_XYZ RunTo Well_XYZ DONE RunToTimeFrame 20 RunToTimeFrame 20 DONE “The automate provides the liquid” RunTo Well_XYZ RunTo Well_XY ERROR RunTo Well_XY not reached until end of experiment RunTo ExperimentEnd RunTo ExperimentEnd DENY No screen is running
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Acquisition 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
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® OLYMPUS LIFE SCIENCE EUROPA GMBH Postfach 10 49 08, 20034, Hamburg, Germany Printed in Germany 2008 12 M 2.1.0.14
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