After performing immunohistochemistry on the FFPE tissues, image the sections using the digital pathology imager. Launch the software to perform spectral unmixing of the annotated images. Select File, then click Open Image, and choose the QPTIFF files to upload the images into the software.
Allow the stamps marked as informed projects to upload into the project. Load the QPTIFF files prepared for the autofluorescence compensation. Select the autofluorescence on the image tool to draw a line on the image from the unstained slide through autofluorescent structures like erythrocytes and collagen to compensate for autofluorescence.
Then navigate to the Edit Markers and Colors section. Assign marker names that match the Opal fluorophore, and adjust the color settings to your preferred option. Select Prepare All located in the lower left corner of the interface to initiate the unmixing of fluorophores.
Examine all images to check the visibility of all signals and the successful completion of the unmixing process. Select the eyeball icon to turn off and on all the markers one by one to check the quality. Now navigate to the Export tab and create a new empty export directory by clicking the Browse button located under the Export directory.
Select composite image and component images multi-image TIFF under the images to export tab. Navigate to the Batch Analysis tab found vertically on the left for batch processing of slides and select create separate directories for each item under the export options. To add slides for analysis, select QPTIFF files under the Add Slides button and load these into the batch analysis.
Select Run to commence the batch processing of slides. Create a new folder containing only the component files from spectral unmixing, ensuring the hierarchical folder structure remains intact. Launch the whole slide viewer software.
Click Create Project on the left side and create or select a new empty folder with an appropriate name. Then click Automate and choose Show script editor. Copy and paste the existing script and modify the location to direct it toward the folder that contains all the slide component files.
Select Run to begin the process of batch stitching slides, and then allow it to complete. Now move the recently generated OME TIFF files into the QuPath project and save them as a new project. When a new window appears, select set image type as fluorescence, then click the Import button.
Navigate to the menu on the left, select a sample from the list and double click to open the chosen sample. Adjust the intensity of the channels by clicking the Contrast icon to enhance visibility. Select all channels and opt to reset.
Ensure the autofluorescence is toggled off. To draw a region of interest or ROI for the tumor, click the contrast icon again then select Show grayscale. After that, select the tumor marker channel and adjust the intensity for optimal visibility.
Click the brush tool to draw an ROI around the tumor. Click the one tool and adjust the ROI while pressing the alt key to smooth the ROI from the outside. When finished, merge any separated parts of the tumor into the same ROI.
Right click the ROI annotation in the list on the left, select Set Properties, and provide a suitable name. For instance, Tumor. Expand the ROI for the invasive margin from the tumor region by selecting Objects, then Annotations, followed by Expand Annotations.
Choose the desired size for the expansion radius. Select remove interior and constrain to parent. Click on the contrast icon, select the autofluorescence channel and adjust the intensity for optimal visibility.
Click the wand and adjust the ROI while pressing the alt key to smooth the ROI from the outside and remove any background that should not be part of this ROI. Right click the annotation in the list on the left. Then select Set Properties to assign a suitable name, such as invasive margin or IM to the ROI.
If desired, change its color. To export the annotations, navigate to the File option, click Object Data, Export as GeoJSON, and select Export All Objects. Continue with the default selection on export as feature collection, and save it in a preferred location.
