- Description of the purpose of each step of the image analysis pipeline along with example outputs
- 1. Correct Illumination Calculate and Apply
- 2. RunStarDist
- 3. OverlayOutlines
- 4. IdentifySecondaryObjects
- 5 IdentifyTertiaryObjects
- 6. IdentifySecondaryObjects
- 7. IdentifyTertiaryObjects
- 8. Threshold
- 9. Maskobjects
- 10. OverlayOutlines
- 11. EnhanceOrSuppressFeatures (Neurite)
- 12. Threshold
- 13. Maskobjects
- 14. OverlayOutlines
- 15. MeasureImageAreaOccupied
- 16. MeasureObjectNeighbors
- 17. ConvertObjectstoImage
- 18. ImageMath (Invert)
- 19. DistanceTransform
- 20. GraytoColor
- 21. OverlayOutlines
- 22. Save Images
- 23. Export to Spreadsheet
Calculates and applies an illumination function to the images to correct for uneven illumination in the images of SMA-staining. The method 'Background' was selected to correct for the high background staining in some of the images of the SMA-stain (Texas Red).
Uses the StarDist algorithm (https://github.com/stardist/stardist) to segment nuclei
Checks the accuracy of the outlines of the nuclei segmented by RunStarDist
Segments the whole cell outlines (for CD8)
Segments cytoplasm (whole cells minus the nuclei) (for CD8)
Segments the whole cell outlines (for SMA)
Segments cytoplasm (whole cells minus the nuclei) (for SMA)
Sets the intensity threshold for deeming cells as marker (CD8) positive
Keeps the thresholded areas of the image using a mask and identify the cells present in those areas
Checks the accuracy of detecting CD8+ cells by overlaying the outlines of the CD8+ cells on the grayscale FITC image
Enhances thin, elongated areas of the image - this was performed to enable detection of the SMA+ fibroblasts
Sets the intensity threshold for deeming cells as marker (SMA) positive
Keeps the thresholded areas of the image using a mask and identifies the cells/nuclei present in those areas
Checks the accuracy of detecting FoxP3+ cells by overlaying the outlines of the SMA+ cells on the grayscale TexasRed image
Measures the area of image occupied by SMA+ cells
Measures the number of CD8+ cells that lie within a radius of 20u, 50u, 100u, and 200u from the SMA+ areas
Coverts the identified CD8+ cells to a binary image for performing further downstream measurements
Inverts the image such that the darkest pixels become the brightest pixels
Computes the distance of a foreground pixel to the nearest background pixel and rescales the resultant image in a way that the largest distance is 1. This step is performed to obtain more nuanced measurements of the distances of CD8+ cells to SMA+ areas.
Creates a composite color image merging the DAPI (DNA), FITC (CD8), and Texas Red (SMA) channels
Overlays the outlines of the CD8+ and SMA+ cells on the composite color image
Saves all the overlays to designated folders
Exports all measurements to a spreadsheet for downstream analysis
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