Collagen quantification with QuPath – Protocol

I have prepared and organised a protocol for Collagen quantification analysis via QuPath. If you want to include other options related to the Collagen quantification leave a comment and I will include the screenshots to update the protocol.

Create a project.

2. Load all the images of the same batch in the project that was just created by dragging them in the project window.

3. Obtain pixel size:

Load the image with the scale bar and open it.
With the line tool, draw a line onto the scale bar.
Click on the image tab and double click on the pixel width value.
Insert the length of the scale bar in the popup window and click “ok”.
Write down the value of the pixel width since it will be used in all other images.
Load image to analyze.

4. On the image tab, double click on the pixel width value and set pixel width and height values.

5. Zoom in on the image and draw a square on a cell (stained in pink/red).

6. Double click on the Stain 1 value in the image tab.

7. Select “yes” in the popup window, rename the Collective name in “MT” and the name of the stain in “Cells” and click “Ok”.

8. Zoom in on the image and draw a square on a blue stained area (collagen).

9. Double click on the Stain 2 value in the image tab.

10. Select “yes” in the popup window, the name of the stain in “Collagen” and click “Ok”.

11. Zoom in on the image and draw a square on a background area (white).

12. Double click on the Background value in the image tab.

13. Select “yes” in the popup window and click “Ok”.

14. Use the closed polygon tool to select an area of the fibrotic capsule (double click to finish the shape).

15. If it is the first image you are analysing, you have to create a Threshold that then will be applied to all other images of the stack:

Click on “Classify → Pixel Classification → Create Thresholder”.
Use the settings below for the thresholder:

16. Following images:

Click on “Classify → Pixel Classification → Load pixel classifier
Set the classifier as shown above and click “Measure”

c. Set the classifier as shown above and click “Ok”

d. The collagen area will be the positive % value on the left that can be copied and pasted in excel.

19) Here is shown the Positive % of collagen quantified within the region of interest

Preprint

Preprint

Collagen quantification with QuPath – Protocol