Selecting and Exporting ROIs from Stack

I have several fluorescence microscopy images with 10+ channels (each representing a different marker) which I would like to analyze in ImageJ.

I have imported the images as a virtual stack and converted to a normal stack via Image > Duplicate. I would like to define ROIs for the entire stack based on thresholding a single marker (one slice in the stack, this is a membrane protein which should define my cells of interest). It appears that Image > Adjust > Threshold applies the same threshold across all slices in the stack. Ideally, I would like to use background subtraction to define ROIs as best as possible.

I would then like to export/save each ROI as a separate image. Is this possible?

Thank-you very much for your guidance!


can you upload an example image? That would help to understand what your goal is.

Since your image fits into your RAM memory you don’t have to load it virtually in first place, you can directly load it “the normal way”.

Can you explain more what you mean with thresholding one slice with a specific marker - do you mean different channels?

By the way, Adjust > Threshold can do thresholding per slice, or for the full stack together (the latter is the more normal way). When you click Apply you can check/uncheck the option Calculate threshold for each image.