Nuclear and cytoplasmic quantitation


#1

I am a new CellProfiler user and I am very impressed with this software. I have a set of images stained with DAB. The cases show both cytoplasmic and nuclear stain. Is it possible to quantify in the same image the nuclear intensity and the percentage of positive nuclei? I am having trouble generating the pipeline.
Any help and guidance will be very much appreciated



#2

Hi,

Glad you found CellProfiler. Yes, you can do measure these, with caveats. If you could add a nuclear marker, that would help. The nuclei appear to be a little blue here (is that only DAB, or is there another marker here like Hematoxylin?). I have created a pipeline for you, assuming that:
(1) The nuclei are blue-ish in hue (and of course the cytoplasm are the typical brown DAB stain color)
(2) When you say, “percentage of positive nuclei”, I am assuming you mean “blue-ish nuclei that also stain darkly for brown/DAB”. Please clarify though, because it is not clear to me how you define “all nuclei” and “positive nuclei”.

Side-note: Avoid JPG images because they are a lossy format. Try to save in, say, TIFF or PNG and no information should be lost via compression.

Look at my attached pipeline.

  • UnmixColors is key for histological images. I added DAB, but also guessed at the blue-ish setting by adding our default Hemaoxylin. Feel free to change this to another setting but you need something to segment the nuclei with
  • IdentifyPrimaryObjects: You will need to change the Threshold Correction Factor almost certainly. I changed this from 1 (default) to 2, but this may vary with staining parameters.
  • IdentifySecondary: This seems to miss some brown regions, but that is because I am working under the model of “Define nuclei as small blue regions, and then grow out from those to the brown cytoplasm”. If there is no blue-ish nucleus defined, there will be no brown Cell object.
  • DisplayDataOnImage: This can be disabled (click the checkbox in the pipeline list) and is only to help you get a sense of the threshold applied in ClassifyObjects
  • FilterObjects: This is only useful if you want to process the positive nuclei downstream (disable otherwise)
  • ClassifyObject: This calculates the percentage positive

Hope that helps, and let us know if you need help in case I made some wrong assumptions.

Cheers,
David
DL_DAB.cppipe (11.9 KB)


#3

Hello David; please find attached file, it is a jpeg ROI from bone marrow stained with DAB stain for nuclear protein. could you please help me as i am a beginner for CP. I have tried your suggested pipeline but either it did not work or even after modifying some parameters, I could n’t get any output results. I am looking to get brown stained nuclei over all nuclei, more additional parameters are also appreciated such as absolute cell density in each tissue section area, large sized cells vs small sized ones (cut off may be lymphocyte size ~8um). also i need out put pseudo colored to verify manually. THANKS IN ADVANCE. Reda Mahfouz, MD, PhD, Cleveland Clinic


#4

Hello Reda,

Have you made progress on this? Sorry for the slow reply – in the interim, we have migrated to a new forum and for whatever reason, your attached image appears quite small. If you would still like help, could you re-attach another image? Thanks!