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How do I clear the gradient of an image across pixels? (Image processing for the intensity of fluorescence)

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I have 200 images, 100 each of two different filters. I averaged them all and ratioed them properly to get one image. However, this image doesn't have a clear gradient across the pixels. My end goal is to analyze the intensity of the fluorescence and graph it. How would I clear the gradient without disrupting the image?
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Bjorn Gustavsson
Bjorn Gustavsson on 1 Nov 2022
It is way easier to give recommendations if you attach/inline the average images and the ratio, and preferably tell us if you've reduced dark-frames and made some kind of flat-field corrections.

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Answers (1)

Constantino Carlos Reyes-Aldasoro
I am afraid the question is not very clear. What do you mean by "doesn't have a clear gradient across the pixels"? This seems to be contrary to "How would I clear the gradient without disrupting the image?" which would suggest that there is a "gradient" in your image.
I guess that what you have is "shading" i.e. the background intensity is not uniform, and you would like to remove and have a uniform background and with that you can analyse the foreground (like your fluorescent objects of interest).
Have a look at this function, it may help if you want to remove the shading:
And the explanation of the algorithm can be found here


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