How can I make the background of a microscopy image perfectly black?

Hi all,I’m new to the forum !
I have a microscope fluorescence image of which the background is not perfectly black. Is there a way I can pick a spot on the image and “tell” ImageJ/FijIJ that it should define this as perfect black, thus adjusting the colours of the entire image the way you can do it in Photoshop ?

I’d be very grateful if anyone could help me. I have tried to search for information on Google and also tried out a lot of functions, but so far I haven’t succeeded. I am using ImageJ 1.50b with the FiJI plugin, if that is of any use.
Thanks !

Welcome to the forum, @Coldplay!

The operation you describe is known as “background correction” or “background illumination correction” and there are many ways of doing it. The best approach will depend on your image—especially the acquisition conditions and modality.

So: can you please post a sample image? And describe how it was generated?


Hi @Coldplay,

A very simple method is:

  1. Look up the value of the spot that you want to be turned black. You can see the value of one pixel in the status bar, or you can measure the mean of a small ROI.

  2. Process > Math > Subtract that value (this reduces the value of every pixel by that amount).

However, as @ctrueden pointed out, this may not be the best approach. Most microscope images have uneven background across the field of view. That’s why ImageJ has more sophisticated options such as the “rolling ball” method, or subtracting a reference image.

If you post a sample image and description, it will help you get the best suggestions.


Two pages linked below might give you some hints.

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