Hello there,

I am pretty new in using ImageJ and have encountered some problems when trying to do colocalization analysis.

I have stained the nucleus of my cells with DAPI and want to colocalize it to another dye (which is obviously not in the nucleus, I just need to prove it with some data.) As far as I know, Pearson’s coefficient should be a negative value, because it is anticolocalized.)

When I tried colocalization with JACoP, I got positive Pearson’s coefficients. I think this is because it takes the whole image with a lot of background into account and not the single cells, that I have selected as ROI.

Therefore, I switched to coloc2, which indeed gives me a negative Pearson’s coefficient.

Still, I struggle with the Mander’s coefficients: When I take my whole image with many cells on it and select one cell as ROI, I get m1 = 0.00 and a m2 = 1.00. (The Pearson’s coefficient is -0.5)

When I crop the image and clear everything outside the ROI, the m1 = 0.349 and the m2 = 0.296. (Pearson’s coefficient is -0.58)

-What is the reason for the strange Mander’s coefficients in the first case?

-Why do Pearson’s and Mander’s coefficients change, although I have selected the exact same ROI?

Thank you very much for your help, I appreciate it very much.