Definiens. Tissue Studio 4.2. Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection

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Claud Lester
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1 Definiens Tissue Studio 4.2 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection

2 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection Imprint and Version Copyright 2015 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 Introduction...5 Load Solution...7 ROI Detection...9 Cellular Analysis Export Run and Review

4 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection 4

5 Introduction Introduction This tutorial will guide you through the steps required to perform an analysis using the manual ROI detection functions of Definiens Tissue Studio in combination with the Vessel Detection function. You will learn how to manually select regions of interest (ROIs) based on an initial segmentation step. We will then submit the detected ROIs to a general vessel detection that identifies vessels and classifies them according to size. In addition, we will show you how to import metadata along with your images into the workspace it is often necessary to have additional image information in order to set up an analysis. For instance, you might want to select your training data so that it spans images from all experimental conditions. Importing metadata into Tissue Studio is straightforward all metadata must be in a CSV file called metadata.csv and be in the same folder as your image files. In order to link images and metadata, metadata.csv must contain a column with the heading image, which contains the unique image names (without file extensions). 1 Start Tissue Studio in the Tissue Studio portal. 2 Select the Load tab and click the Import Folders button. The image files you need are located in 2B_BF_Slides_Vessels\images. Select Aperio ScanScope with resolution as the scanner type. 3 Type a name for the workspace and browse to the location where you want to save it (use the prefix WS_ to distinguish the workspace folder easily from other folders in the same directory). Press Finish. The metadata contained in the file metadata.csv are automatically loaded into the workspace. This is highlighted by the link in the Project Properties window. 4 Select all four slides in the root Workspace folder and create a training data set with the Training Data button. 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 3: Metadata Import, Manual ROI Selection and Vessel Detection 6

7 Load Solution Load Solution We now want to manually define the ROIs to be analyzed with an initial segmentation step. The manual classification result must first be defined (and saved) for every image individually; we can later execute this solution in batch mode. 1 In the Workflow window switch to the Configure tab. 2 Press the Load Solution button. 3 In the Load Solution dialog box, select the solution Draw Polygons > Vessels and confirm with OK. However, we would like to use the action Manual ROI Selection (Select Segments) instead of Manual ROI Selection (Draw Polygons). 4 Delete the action Manual ROI Selection (Draw Polygons) by clicking on the minus sign. 5 Click on the plus sign of the red group and add action ROI Selection (Select Segments). 7

8 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection 8

ROI Detection ROI Detection For training purposes, we want to select the central tissue region in all four images.

2 Configure the settings according to the analysis task: Use metadata from file should be active, and select IHC Brown chromogen and Cytoplasm under Staining Information.

You can modify individual objects through reseg- Figure 1: General Settings action mentation or manual cutting.

9 ROI Detection ROI Detection For training purposes, we want to select the central tissue region in all four images. 1 Click on the General Settings action (Figure 1) to activate its widgets (the active action is indicated by a yellow frame). 2 Configure the settings according to the analysis task: Use metadata from file should be active, and select IHC Brown chromogen and Cytoplasm under Staining Information. 3 Change to the Manual ROI Selection (Select Segments) action. Set the Segmentation Scale to 8 and press the Segmentation button (see Figure 2 on page 10). You can modify individual objects through reseg- Figure 1: General Settings action mentation or manual cutting. 4 To achieve a finer segmentation scale, click the Resegment Object button, reduce the Segmentation Scale and click on the object to be resegmented. 5 Alternatively, you can simply cut out your region of interest in a larger object by using the Cut Manually button and using the mouse to cut out an object from a larger object (for more details see the Tissue Studio User Guide). 6 Activate one of the ROI buttons and click on the objects you want to select (see Figure 3 on page 10, left). 7 Activate another ROI class and press Classify Remains to classify remaining objects within their respective classes you can edit class names and colors using the Edit Classes buttons. 8 Press the Merge button to merge all objects of the same class (see Figure 3 on page 10, right). 9 Press the Confirm button to save the selection. NOTE: You can also apply manually selected regions inside other workspaces. Copy your workspace using File > Save Workspace to another location and copy the ManualClassifications folder to the new workspace folder. The classification is used when the Load button in the Manual ROI Selection action is pressed. 9

folder and can be reapplied to the images with the Load button in the Confirmation section (in case you close the program

10 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection Figure 2: Segmentation result. CD_34 (bottom, right) has been segmented using the Cut Manually function The manual annotations are saved in the workspace folder and can be reapplied to the images with the Load button in the Confirmation section (in case you close the program before the configuration of your solution is finished). Figure 3: Results for manual classification (left) and final classification (right) 10

4 Activate the Select button and choose eight subsets on all slides (see Figure 5). Figure 4: ROI selection for cellular analysis Up to 12 subsets can be selected.

11 Cellular Analysis Cellular Analysis 1 Activate the Initialize Cellular Analysis action. 2 Select the classes you have used to create the polygons to be submitted to the analysis by deselecting classes you don t need. 3 Set the Magnification to 5x. 4 Activate the Select button and choose eight subsets on all slides (see Figure 5). Figure 4: ROI selection for cellular analysis Up to 12 subsets can be selected. 5 Switch to the Vessel Detection action. 6 Set the IHC Threshold to 0.35, Min Stain Area and Gap to close to 0 and press Preview Settings to see the vessel stain region (see Figure 6 on page 12, left). On closer inspection, you may notice that some lumen areas are not yet recognized this is because these lumina are not entirely enclosed by the vessel wall. You may also want to exclude small bits and pieces that should not count as vessels. 7 Set the Gap to close to about 5µm to close small holes in the vessel wall (see Figure 6, right). 8 Set Min Stain Area to a value larger than zero to exclude stain fragments of the specified size. Figure 5: Subset selection 11

Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection Figure 6: Preview of vessel settings with Gap to close set to 0µm (left) and 5µm (right) Here you should only exclude small

9 Press Preview to detect vessels on an upper level (vessels are classified as vessel with lumen and vessel without lumen ) and vessel lumen and vessel wall on a lower level.

12 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection Figure 6: Preview of vessel settings with Gap to close set to 0µm (left) and 5µm (right) Here you should only exclude small fragments and artifacts. With the Vessel Classification action, you can then exclude entire vessels from the analysis if necessary. 9 Press Preview to detect vessels on an upper level (vessels are classified as vessel with lumen and vessel without lumen ) and vessel lumen and vessel wall on a lower level. 10 Use the up and down arrows in the main toolbar to switch between levels (see Figure 7). You can open the Scene Information pane (View > Scene Information) to display some useful statistics for the currently active window and for all subsets (see Figure 8). 11 Switch to the Vessel Classification. 12 Select the feature Area (µm²) and keep the default settings. 13 To exclude small vessels from the analysis activate the Use exclusion checkbox and click on the button at the right side of the Feature 1 panel. 14 In the opening Select Single Feature dialog choose Object Features > Geometry > Extent > Area (µm²) and confirm with OK. 15 Set the Operator to < and assign a value of Press Preview to classify the vessels into Vessel Large, Vessel Medium or Vessel Figure 7: Vessel detection on lower (left) and upper (right) levels 12

13 Cellular Analysis Figure 8: Scene Information window for Vessel Detection action Small and exclude vessels with an area smaller than 50µm (see Figure 9). Figure 9: Vessel Classification result 13

14 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection 14

Export Export The Default Export action takes screenshots of the classification results and creates result files with all information about the identified ROIs plus the

2 Choose the screenshots you want to retrieve and make sure the Statistics and Statistics Per Slide checkboxes are active.

15 Export Export The Default Export action takes screenshots of the classification results and creates result files with all information about the identified ROIs plus the nucleus objects and marker area within the ROIs. 1 Click on the Default Export button. 2 Choose the screenshots you want to retrieve and make sure the Statistics and Statistics Per Slide checkboxes are active. TIP: If you want to reuse the solution, we recommend storing it under the following paths:..\application Data\Definiens\TissueStudio\Data\Solutions (Windows XP) or C:\ProgramData\Definiens\TissueStudio\Data\Solutions (Windows 7). This way it is directly accessible via the Load Solution button. 15

16 Tutorial 3: Metadata Import, Manual ROI Selection and Vessel Detection Figure 10: The Review Heat Map window 16

17 Run and Review Run and Review Batch processing is always performed using the currently loaded solution. When you run the 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 Select the images you want to submit to the batch analysis and click Analyze Selected Images or simply click Analyze All Images if you would like to get results for all images in the workspace. 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 done, switch to the Review tab. 4 Click on the Open Results Folder button. Here you will find all exported results (screenshots, statistics and solution.dax file). You can also review slides in the Review Heat Map window (Figure 10). Select any ROI and press the Details button. 5 Open the Review Heat Map via the View menu.. Thumbnail view is visible by default; you will need to select the name of the result container in the drop-down box (for information on result containers, see the Tissue Studio User Guide). 6 Choose the Level, Feature and Class via their respective drop-down boxes. You can additionally choose statistical features. 7 Adjust the slider to change the thumbnail transparency and adjust the values for minimum or maximum at either side of the color scale. Press Export to export the image. In the example in Figure 10, we have highlighted the median areas of vessels in the Review Heat Map. The largest vessels are towards the red end of the spectrum; the smallest are towards the blue end. 17

18 Corporate Headquarters Definiens AG Bernhard-Wicki-Strasse Munich Germany Tel Americas Headquarters Definiens Inc Aston Avenue, Suite 190 Carlsbad, CA 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.

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

Definiens. Tissue Studio 4.4. Tutorial 4: Manual ROI Selection and Marker Area Detection Definiens Tissue Studio 4.4 Tutorial 4: Manual ROI Selection and Marker Area Detection Tutorial 4: Manual ROI Selection and Marker Area Detection Imprint and Version Copyright 2017 Definiens AG. All rights