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I am trying to measure the number and/or intensity of puncta in one channel that do not overlap with a second channel. I have two channels that largely overlap, but one has puncta that are outside the area of overlap. I would like to quantify these in some way (or even better, in multiple ways). For simplicity you can think of a green box that overlaps with a red box, surrounded by green dots and I want to count the dots and measure their intensity. My images have a dark background and are in a stack.
What I think I want to do is this:
1. Generate a mask of the red channel using the light background setting to exclude the area of red signal (I have figured this step out)
2. Convert that mask to an ROI (not sure how to do this part)
3. Apply the ROI to my green channel (if the ROI shows up in the ROI manager, I know how to do this)
4. Measure the intensity inside the ROI (I know how to do this)
5. Count the number of particles (I have a general idea of this, but not the specifics)
My questions are
1. Will this work across stacks or do I need to generate a projection first?-I'm assuming for the particle counting, a projection would be better (I haven't had much luck with using 3D Object Counter in the past)
2. Is it possible to convert a mask to an ROI, and, if so, how?
Also, if you have suggestions about an easier/better way to make these measurements, I would love to hear them.
Tips for quantifying puncta would also be useful.
Be warned: I am fairly inexperienced with ImageJ and not excessively computer savvy. I am really only comfortable using the existing features of ImageJ and its plugins. (I have Fiji)
Thanks!
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