I have done a fluorescent stain and I want to count the number of cells that have the stain colocalized in the nucleus. However, there are many more cells stained than are the ones that I care about. In the sample image, many cells are stained red, but I only want to know how many green cells also have the red stain.
Most methods for automatic cell counting online simply count all of the cells in the image at once, but I can't do that because I only want the ones that are in the green cells. Additionally, my cells are in a z-stack, I only show one plane in the example image here.
I can't do a colocalization analysis because my green stain is cytoplasmic and the red is nuclear.
Is there a way to do this analysis automatically? Or do I need to count this manually? If I do need to do it manually, any suggestions on how?
Thanks!
Dear Rebecca!
You can count the number of cells with green fluorescence using the ImageJ program.
https://imagej.nih.gov/ij/download.html
I am not sure I understand your description of the problem. In the beginning, you write " I want to count the number of cells that have the stain colocalized in the nucleus", but later, it is stated that "I can't do a colocalization analysis because my green stain is cytoplasmic and the red is nuclear." This is a bit confusing. If I understand well, you want to see how many of the cells with the red nucleus have a green cytoplasm, no? I agree with the above answer, Image J is great for this sort of thing, but I want to make sure I understand exactly what you are trying to do.
Hi Rebecca,
If I understood your question correctly what you would like to do is take the green cells only, and then count how many of them have a red nucleus. As others have suggested previously, ImageJ would be a great way to deal with this problem and, if you are dealing with a lot of images, you can also create a macro within ImageJ that allows for the quantification to happen for all images automatically.
To give you something a bit more tangible, here is an easy to follow approach to your problem:
It may not be the most robust way, but it should be fairly intuitive. If you want to automate the process you can have a look at my github for some pointers.
https://github.com/emltwc/EnteroEndocrine/blob/master/andre_quant.ijm
Best wishes,
Máté
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