Definiens. Tissue Studio 4.4. Tutorial 4: Manual ROI Selection and Marker Area Detection

Transcription

1 Definiens Tissue Studio 4.4 Tutorial 4: Manual ROI Selection and Marker Area Detection

2 Tutorial 4: Manual ROI Selection and Marker Area Detection Imprint and Version Copyright 2017 Definiens AG. All rights reserved. This document may be copied and printed only in accordance with the terms of the Frame License Agreement for End Users of the related Definiens software. 2

3 Contents Contents Getting Started...5 General Settings...9 Manual ROI Detection Nucleus Detection Marker Area Detection Export Run and Review

4 Tutorial 4: Manual ROI Selection and Marker Area Detection 4

5 Getting Started Getting Started This tutorial will guide you through the steps required to perform an analysis using the manual ROI detection function of Definiens Tissue Studio IF, in combination with the Marker Area Detection function. You will learn how to manually select regions of interest (ROIs) based on freehand drawing. Finally, the detected ROIs will be submitted to a general marker area detection. Start the program in the Tissue Studio IF portal (if you do not select any portal, the software will automatically start after three seconds with the portal you selected during the last session). When working with fluorescence images, the different channels are often available as independent image files. In order to combine these files into one project, the import must be customized. 1 Go to File > Customized Import to launch the Create New Workspace dialog. Name the workspace WS_example2C and save it to a convenient location (we recommend using the prefix WS_ to distinguish the workspace folder easily from other folders in the same directory). Confirm with OK. 2 In the opening Customized Import dialog make sure the Workspace tab is active and name the import Example2C in the Import Name field. 3 In the Root Folder field, press Select and browse to the image folder of this tutorial. In the Master File field, select the first image file Mouse_DAPI.tif Customized Import uses the file header information to assign each file to the right project. In this example, the first part is the name of the tissue source or, in other words, the name of the project or scene. The second half of the file name indicates the biomarker, which is synonymous with the chan- NOTE: This book assumes you have already worked through tutorial #1 (Composer and Nuclear Markers) and are familiar with the basic functions and workflow of Tissue Studio. You may also want to read chapters 1 3 of the Tissue Studio User Guide. This tutorial is intended to be an example and will not explain every aspect of the software. For more information refer to the User Guide. 5

6 Tutorial 4: Manual ROI Selection and Marker Area Detection Figure 1: Settings for the Customized Import dialog nel or layer information. To import the images accordingly you have to use search string blocks, which are treated as variables. 4 Change the Search String field to {{root}\{scene}_{layer}.tif:reverse} You can save time typin g new variables by placing the cursor in the field and selecting them from the Insert Block drop-down list. 5 Keep the Scene Name as {scene} to automatically label the projects according to the information from the file names. 6 Click the button Test to check in the Preview field whether the import template is correct or not. If correct, the fields will appear in the Preview pane. If any field is incorrect you will get an error message. 7 Click on the Save button to save the import template (.xml) for further use. The.xml import template is automatically stored in the user s application data folder..\appdata\roaming under Definiens\3.6.x\import. From now on the customized import template is available in the list of the predefined import templates when using the Import File or Import Folder buttons. 6

7 Getting Started Figure 2: Multiple layer view of the imported image file (Tissue Slide.Mouse) More detailed information on how to use the Customized Import is described in the Tissue Studio User Guide under Help in the main menu. 8 Start the import by clicking the OK button. 9 Double-click on one of the two loaded images to open it. The images show a pancreatic islet both in mouse and rat tissue with label- Figure 3: Settings for the Image Layer Mixing dialog 7

8 Tutorial 4: Manual ROI Selection and Marker Area Detection Figure 4: Image before and after equalization ing for insulin (beta cells), glucagon (alpha cells) and somatostatin (delta cells) plus DAPI for the nuclei. By default only three layers are displayed in the image. 10 To display all four layers go to View > Image Layer Mixing. In the Image Layer Mixing dialog choose the colors R and B for Layer In the drop-down menu under Equalizing choose Linear 1% to enhance the image display. Confirm with OK. Create a Solution 1 Switch to the Configure tab and press the New Solution button 2 To add or delete actions, you can press the plus and minus signs. Use the arrows to move the actions up and down. 3 As ROI Detection add Manual ROI Selection (Draw Polygons). 4 As Celluar Analysis add Nucleus Detection and Marker Area Detection. 5 The respective actions are now assembled in the Analysis Builder window. You can now configure the solution. 8

9 General Settings General Settings If your imported images contain no metadata, you will need to manually set the magnification and resolution based on the scanner properties. Correct values for these parameters are crucial to your analysis, as all subsequent calculations are based on them. 1 Set Magnification to 40x and Resolution to 0.2µm. You can rename the imported layers (channels) with the Edit Names button. 2 To decide which layer is related to which biomarker information, split the image information by clicking on the button to the very right of the main toolbar or right-clicking into one of the images, and choosing Single Layer Display from the context menu. 3 Browse through the four separated layers using the buttons now visible to the right of the toolbar. You can see the Layer 1 is DAPI, Layer 2 is Glucagon, Layer 3 is Insulin and Layer 4 is Somatostatin. 4 Press the Edit Names button to launch the Edit Layer Names dialog. Change the names of the layers in the right-hand column and confirm with OK. The altered name of the active layer is now displayed in the Status bar at the bottom of the window. 5 Repeat the second step to go back to multiple layer mixing mode. Figure 5: Edit Layer Names dialog 9

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11 Manual ROI Detection Manual ROI Detection 6 Activate the action Manual ROI Detection (Draw Polygons) in the Analysis Builder. The manual annotation should include the entire pancreatic islet area in both images indicated by the blue stain (insulin). 7 Ensure Use Magnetic Snap is unchecked and the Editing Type is set to Add New Polygons. 8 Click on any of the eight ROI buttons to activate the drawing mode (this will also launch a Help dialog). 9 Hold down the mouse button and draw a polygon that roughly surrounds the islet area. Press Confirm when you are happy with the result. 10 Switch to the second image with the Workspace Navigation buttons. Repeat the ROI annotation with the same class (here ROI 1) on the second image. Go back to the first image to proceed with the next chapter. Figure 6: Top left: freehand polygon around the ROI in the mouse tissue image (detail is enlarged for clarity) 11

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13 Nucleus Detection Nucleus Detection 1 Switch to the action Initialize Cellular Analysis. As the images are relatively small we can run the detailed analysis on the full 40x resolution without significantly affecting performance. 2 Mark the ROI class you have used for the manual annotation (here: ROI 1). Set Magnification to 40x and press the Select button to split the image into Background and All ROI. To view the classification, use the button in the main toolbar to show or hide the classification. 3 Switch to Nucleus Detection. In general, four parameter are taken into account for the detection of nuclei: the layer with the nucleus stain information, the layer with the membrane stain to mask regions where no nuclei should be found, a grayscale threshold and the approximate size of each nucleus. 4 Select the layer DAPI in the drop-down list for the Nucleus Layer. Leave the Membrane Mask layer as <not assigned>. 5 Switch to Single Layer Display (via the right-click menu or the main toolbar) and browse to the layer DAPI for easier determination of the nucleus threshold 6 Keep the default Stain Threshold and press Preview Thresholds to view the result. Repeat until you are satisfied with the coverage of nucleus regions (here: 0.4). Remember Stain Threshold is internally referring to the set nucleus size. Figure 7: ROI area available for detailed analysis (image classification is turned on) 13

14 Tutorial 4: Manual ROI Selection and Marker Area Detection Therefore the preview will change according to the changes made to this size. We recommend making an initial estimate of the stain threshold, then defining the nucleus size, before making any final adjustments to the threshold. 7 Click the Select Samples button and choose 1 3 objects that represent larger-sized nuclei. 8 Click again on an object to remove it from the sample collection. The mean size of the sampled nuclei is automatically transferred to the Typical Nucleus Size field. You can also adjust the threshold manually (here 40). 9 Press the Preview button to execute the nucleus detection. You can export two screenshots of this result: one of the original image and one with outlines and an overlay of the result. You can store the view settings manually as they are supposed to be exported. 10 Activate the Export a Screenshot checkbox. The second and third button in the toolbar above the image can be used to adjust the view settings. The second button hides or displays outlines, the Figure 8: Final nucleus detection result (image classification is turned on) 14

15 Nucleus Detection third button hides or displays the classification color. Furthermore you can decide whether you want to display all image layers or only one. 11 Change the view settings according to your needs (here: single layer display of DAPI layer, outlines displayed, classification displayed). 12 Click on Save Settings to store the view settings for export. If needed, you can use the Nucleus Classification action to exclude objects such as very small nuclei and fragments. 15

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17 Marker Area Detection Marker Area Detection 1 Switch to the Marker Area Detection action. 2 Select the somatostatin and glucagon layers in the Marker Detection Settings field. To check for every single marker whether the classification result is accurate, you can activate one marker after the other in the Preview field and then execute with the Preview button. 3 Display the layer Somatostatin by using the layer buttons in the main toolbar. 4 Activate the Preview Marker 1 checkbox only for and press Preview. You will see that the threshold is too high and does not detect the entire signal. 5 Adjust the Somatostatin threshold until you are happy with the result (here 50), then uncheck Preview Marker 1. Remember you are still in the enhanced image view, which is why you may have the impression that not the entire signal region is covered by the classification color. You can change the view settings by opening the Image Layer Mixing dialog again with View > Image Layer Mixing in the main menu and setting Equalizing to None. Thresholds will also differ depending on the analysis task and the user judgment. 6 In Image View, switch to the glucagon layer and check Preview Marker 2 only. Press Preview to check the classification result. Adjust the Glucagon threshold as necessary (here 70). 7 Check both Preview Marker checkboxes and press Preview again. 8 Go back to multiple layer view (uncheck Single Layer Display via the right-click Figure 9: Classifications (1) Somatostatin layer with too high a threshold, (2) Glucagon layer with threshold set to 70, (3) Final classification result for both markers 17

18 Tutorial 4: Manual ROI Selection and Marker Area Detection menu or use the button in the main toolbar) to show the final result on the original image. With the up and down arrow buttons in the main toolbar, you can switch between the NucleusLevel and the MarkerLevel. 9 To export screenshots activate the checkbox in the Export a Screenshot section. 10 Set the view settings as you would like to export them (in this example, three-layer display, outlines hidden, classification displayed) and press the Save Settings button to store the result. 18

19 Export Export 1 Switch to the Default Export action. 2 Keep the default settings for Cellular Analysis. 3 You can configure the view for the export screenshot: Select relevant layers and overlays and click on Save Settings to save this configuration. 4 Make sure that Statistics is activated. 19

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21 Run and Review Run and Review When you run an analysis, a read-only.dax file of the solution with the date and time of the analysis will be stored in the results folder. 1 Switch to the Run tab. In the Workspace window, click on the folder Tissue Slides to display the image list. 2 Click Analyze all Images. You can now follow the status of the analysis in the Workspace window projects will switch from created to waiting to processing and finally to processed or in the Job Scheduler (for information on how to use the Job Scheduler, refer to the Definiens XD Installation and Administration Guide). 3 When the analysis is finished, switch to the Review tab and click the Open Results Folder button Here you will find all exported results (screenshots, statistics and solution.dax file). The Statistics folder the CellularAnalysis_DefaultExport_per_Image.csv file includes not only the data for each marker area individually but also the calculations for any possible overlap of the two (both Area and Intensity). The file ROIDetection_DefaultExport_Per_Image.csv includes values related to the ROI detection, such as the size and number of different ROIs. General scene properties such as the magnification and resolution of the image, and the name of the solution file that was used for the analysis, are in the file AnalysisReport.csv. All parameters such as slider settings that were used for the analysis are included in Config.csv. 4 In addition, you can analyze selected features in the Review Heat Map. To export this view as a screenshot, press the Export button. 21

22 Corporate Headquarters Definiens AG Bernhard-Wicki-Strasse Munich Germany Tel Americas Headquarters Definiens Inc. 125 Cambridge Park Drive, Suite 300 Cambridge, MA USA Tel Definiens AG. All rights reserved. Definiens, Definiens Cognition Network Technology, Tissue Studio, Image Miner, TissueExplorer and Tissue Phenomics are registered trademarks of Definiens AG. All trademarks are property of their respective owners. The information in this document is subject to change without notice and should not be construed as a commitment by Definiens AG. Definiens AG assumes no responsibility for any errors that may appear in this document. For research use only. Not for use in diagnostic procedures.