Processed Composite.tif (15.6 MB)
Hi ImageJ community!
I KNOW that this question falls in line with many questions titled “double positive count” or some variation of that. However, exploring those options has given me data that isn’t what I am looking for.
There are two stains, one for microglia (channel 2) and one for CD68 (channel 1) in tissue (Processed Composite.tif). I am trying to quantify is the number of microglia that are CD68 positive, however CD68 staining is punctuate and there are multiple points of signal inside and outside of the microglia cell body. Using Fiji, I was able to create binary masks of the two channels and did Image Calculator>multiplication function of the two binary masks. However, from this function my resulting image is just the CD58 signal that is inside the microglia (which would give me a count of the number of CD68 puncta are inside the microglia, but not the number of microglia that are have CD68 staining). This result makes sense since the “white pixels” (0 value) are multiplying out the black signal (255) of the microglia binary mask, leaving only the CD68 positive signal within the microglia mask. Example pictures below.
I next tried to make each mask a color LUT (rather than inverted grey LUT) and merged the masks to try and see if color threshold for yellow and green cells only (green=microglia and yellow=CD68 + signal inside the microglia mask) would be the best solution, however I couldn’t figure out how to use this to my advantage.
Now I am currently trying QuPath, using the pixel classifier to threshold my two channels, make annotations of both (based on different threshold parameters) and now I am stuck at classifying these annotations so that I can quantify only the microglia annotations that have the CD68 annotations are counted.
I am curious whether I have truly exhausted working in Fiji (using image calculator) or whether QuPath may be the a better way. I have looked at QuPath’s website and explored their multiplexing and pixel classification tutorials, however was not able to figure out how to work with the different classifications I had created and do “annotation operations” like the union of two annotations or the subtraction of two annotations.
Any input would be greatly appreciated!