Hi All, I have many breast tissue samples and have stained them with two chromogens (Red and Yellow) and there is co-localization areas (orange) that I am interested to quantify as well. What would be the best approach to quantify these three colors intensity in each sample?
Thank you in advance
a straightforward way is to use the threshold option in ImageJ. within this tool there are multiple ways to analyze, depending on what your image is, but this webpage should be a good starting point. for instance, signal area vs intensity
I have used the split channels option to analyze channels separately and then compare signal area
https://imagej.net/imaging/colocalization-analysis
Sorry for asking: does IHC mean that you have a brightfield RGB image with one channel or a multichannel image? If the latter is true, I agree with Molly Brady. But for the other case you would need to do color deconvolution instead of splitting the channels.
Fiji has a number of predefined color deconvolution vectors (Image --> Color --> Color deconvolution). Since I don't know your staining, I don't know if there is one working for you.
Alternatively there is the plugin Color Deconvolution 2, which offers some extra features: https://academic.oup.com/bioinformatics/article/37/10/1485/5913390?guestAccessKey=148a8a4b-24f8-4b42-8742-e985535c9410
And there are of course also other programms, such as Qupath, where you manually annotate cells of interest for automated analysis of the whole sample: https://qupath.readthedocs.io/en/stable/docs/tutorials/cell_detection.html
Hi Sepideh Soukhtehzari ,
I recommand the two posts listed above (by Molly Brady and Suckert Theresa) on the use of the ImageJ's split channels options.
Ludovic Dumont, Ph.D.
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