I have a few confocal stacks that I need to analyze for a research project. I want to essentially measure the fluorescence intensities of cells in one channel using the “measure” command via hand drawn contours. However, there is some non-specific labeling of the antibody I used near the top of the confocal stack, which is not due to the antibody binding to the protein of interest. I found a way to remove this non-specific labeling by doing some fancy work with the “Image Calculator”, since another channel also co-labels the same cells. With the second channel, I subtract the first (generating a new channel with everything BUT the protein of interest), which I then subtract from the original channel.
Since the final channel (after the “Image Calculations”) is what I will be measuring intensities from for publication, I need to know precisely what the “Image Calculator” does. On the documentation for ImageJ, it simply states that an image calculator subtraction is Image = Image1 - Image2, but that doesn’t give me enough information for a publication. Is it literally subtracting the intensity values per pixel from image1 and image2? Or is it removing any pixels that are fluorescing in one image but not in the other according to some threshold? Wether it is doing the former vs. the latter determines whether I need to come up with a new analytical strategy or not.
If anyone has any deeper understanding of the image calculator I would be most appreciative. The other other option for me is to manually look into the code. Thanks!