Bleaching Correction

Hello everyone
I am postdoc who study the odor response of insect antennal lobe.

I use calcium imaging to observe which part of the antennal lobe could be activated by a certain odorant. I choose calcium green which activated by 475nm light as the indicator. Calcium green face to a strong bleaching thus I need to do bleach correction.

Current I use fiji.

Image>adjust>bleach correction–Exponential Fit.

Then I calculate the △F/F0 to know the intensity of the frame.

I read the instruction of bleach correction–exponential fit, it is said that “since bleaching is often not mono-exponential, quantification of fluorescence intensities after bleach correction is not possible. This plugin should only be used to enhance time-course movies for presentation rather than quantification”.

I want to know how to analysis the data when I want to quantify the intensity of the fluorescence also need to do the bleach correction? It seems that bleach correction might bring errors into the data.

Thank you for reading my question.


Hi Wei,
Most of us can only help if we can see what you are working with. Any sample image will do, if you reply to this message you should be able to then upload a sample. It is generally not allowed on a first post.

I am reviving this topic because I am currently compiling some best practice suggestions for various situations in which intensities have to be quantified. Fluorescent live cell imaging does often suffer from bleaching but still a very common readout is fluorophore intensity rather than shape of a structure, so it is an important topic. I am having trouble finding guidelines about this situation however.

Is the current standard still to refrain from post-imaging bleach-correction when measuring intensity? (If preventing bleaching isn’t an option?)

Or is there a sort-of practical ranking which bleach-correction would be least harmful for subsequently measuring intensity? Simple ratio vs. Exponential vs. Histogram matching vs. something even better?

If you have some suggestions that would be fantastic :slight_smile: