Removing overlapping fluorescence from two channel images

I have two-channel image stacks in red and green that consist of large fluorescent protein structures (red) and fluorescent bacteria (green). This work is subject to a patent and I’m not able to paste the images here. However, the question is pretty straightforward.

The green channel only shows the bacteria. The red channel shows the protein and the bacteria because the bacteria are autofluorescent over a wide emission range. What I would like to do is the following:

If there is overlap between the red channel (protein) and the green channel (bacteria), convert overlapping pixels in the red channel to black pixels. This way I can remove bacteria from images where I just want to show the protein. Is there a plugin to do this, or a simple macro I can use?

If the two channels are linearly related, you can use linear unmixing. In the simplest case, find the ratio of red to green in your bacterial pixels, then use Process-> Image Calculator to
Green channel - Red channel *constant = Unmixed Green.

I am sure there are many ways to do this (Zen has them build in), but that is one fairly easy option with FIJI.

Thanks, I’ll give that a try tonight and let you know if I have any issues.

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Hi @kmchlsn2,

In addition to what @Research_Associate said (which is the most elegant, but sometimes it is hard to find the good constant), you can segment your bacteria on the green channel and and use it as a mask to fill in black on the red channel. But this will work only if there is no spatial overlaping between bacteria and protein.

Nico