Sirius red/fast green staining


Hello Lovely People,

I started using the current CP for quantification of collagen area(red area) in hearts and kidney tissue, but I have some difficulties and most the pipelines in the forum does not seem to work in the current version 3.1.8.
Below are sample images (unfortunately cannot be uploaded). Can someone please help me out with a suitable pipeline to build on in a step-wise approach.
Much appreciated


Please find the link to the images below,


Hi there!
You probably would want to use three steps (after loading your images into CellProfiler using the first 4 default modules, Images, NamesAndTypes, etc):

  1. UnmixColors to isolate the red regions. ColorToGray may also do the trick if it’s a nicely separable stain.
  2. Threshold to decide what counts as stained vs not stained. There are many options for this module.
  3. MeasureImageAreaOccupied
    Give it a try!

Hi Amponsah

Continuing on with @AnneCarpenter.

  1. UnmixColor extract the fast red stain

  2. Identify what is not tissue and mask those pixels

  3. Then identify what is red stained. Change threshold correction value to be more or less stringent with the OTSU automatic threshold

  4. Measure Image Area Occupied. Collagen = 9.9% of total tissue

Cellprofiler v3.5.1 Collagen.cpproj (646.8 KB)


1 Like

Thanks Anna much appreciated


Much appreciated Lee but a quick follow up: with regards to the identifyprimaryobject module;

What do you consider/identify as Not tissue and how do you determine its diameter in pixels and also same for the diameter of red stain since they are spread over the tissue.

Many thanks


Hi Amponash

This very much depends on your images. If you follow the pipeline you can see that anything with a diameter from 200pixels to (the entire size of the image) allows you to identify most of the non-tissue areas. I would keep a minimum size because you don’t want to remove very small non-tissue regions as they may be close to objects you wish to count, especially if you are using the “remove if touching edge”. For the diameter of the red stain you can see I did not put a size restriction as there is no need for the question you are asking. This means that even single pixels that pass your threshold will be identified as collagen. This is because you want to know the proportion of tissue that is collagen.

I hope this helps.