How to quantify spots from in situ?


I would like to quantify colorectal cancer stained with DAB staining with RNAscope in situ. But I am not familiar with Cellprofiler, so I wonder if it is the right tool for me and which pipelines would be suitable for doing this. The staining looks like this:®-20-hd-assay-brown-manual-assay

Thanks in advance!


Hello Carl,

Those examples you linked looks “feasible” for CellProfiler. The general idea :

  • First use module “UmixColors” to extract the brown spots.
  • Then use “EnhanceOrSuppressFeatures” to enhance the appearance of the spots and suppress every other unwanted details
  • Then use “IdentifyPrimaryObjects” as usual.

You may find some hints in this example



Hello Minh!

Thank you for your reply! I have started experimenting and it seems promising. I have some issue since I would like to be able to choose spots with the highest intensity since there are some background that is counted as spots after unmix. This gets better if I initially use an image run through color deconvolution with Fiji.
Is it possible to adjust the intensity to only count the highest intensity spots?

Kind regards



It may be easier to first let it count all the spots. Then add MeasureObjectIntensity for the spot objects, then apply FilterObject in which you can freely choose a threshold of intensity to filter the unwanted spots.

This way you can easily re-visit and adjust cutoff threshold without repeating a whole process.

Hope that helps.


Hi Carl,
You can also try using one of the Background/Robust Background threshold methods- they’re optimized for only picking out the very brightest areas of an image.