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

Hello,

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?

[Bjorn Gustavsson]

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.

Answer 1

[Constantino Carlos Reyes-Aldasoro]

Hello

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:

https://uk.mathworks.com/matlabcentral/fileexchange/68333-shading-correction?tab=reviews%2F2069223

And the explanation of the algorithm can be found here

https://digital-library.theiet.org/content/journals/10.1049/el.2009.0320