Hello, everyone.
Recently, I used the fluorescence microscope to measure R-GECO1 (calcium sensor) signal. About 120 pictures were recorded in 25 min. But I found these pictures has the problem of photobleaching. Now, I would like to correct the bleaching using imageJ (Fiji). I found Fiji has the function for correcting bleaching (Image/Adjust/Bleaching correction). However, I read the manual of methods (there are there sub-methods), the authors said “This plugin should only be used to enhance time-course movies for presentation rather than quantification”. The question is, that anyone knows the method can be used for correcting bleaching, and then the corrected result can be used for further quantify the fluorescent intensity? Thank you very much!!!
Shouguang
I think you can refer to the S2 of this paper to test the bleaching rate at your Laser power.
Maria Ott. et. al., Single-Particle Tracking Reveals Switching of the HIV Fusion Peptide between Two Diffusive Modes in Membranes, J. Phys. Chem. B, 117, 13308−13321(2013).
Hi, jinghe, thank you for your answer. I read the S2 of the paper. I think I will test the bleaching rate of the lamp power next time. But do you know any method can be used for solving my data now? Thanks.
If you have gotten the bleaching rate, the data can be corrected by programming by yourself.
I don't think it is good idea to use math for solving the problem with photo-bleaching. Math is only reliable if you definitely know that the equations are linear which is not the case with many biological problems.
Rather I suggest you use a good photo-bleaching preventing agent, basically anti-oxidants which should maintain your fluorescence signal. Try to use low intensity for the illumination and open the aperture if you are using a confocal. If you are using something like FITC, making the mounting media alkaline such as a pH of 8 or 9 really helps. I like the Mowoil mounting media with some dabco in it, I have images them for 'years' without much photo-bleaching.
Regards
We routinely do bleach correction before quantifying 4D confocal data. It works fine as long as the signal doesn't bleach to the point where you have lost significant information.
That plugin should produce a curve fit to your data, so you can judge by eye whether the correction is likely to be accurate.
Thank you for your answer, Nurul. Photo-bleaching preventing agent is an ideal method, I think. I will try to use them next time. By the way, do you know which anti-oxidant agent is the best for plant when performing fluorescent experiment? Thank you!
Hi, Benjamin, I used the method of "exponential fit" to correct the bleaching. Most of stacks showed a very nice fitting, R^2 >=0.95. So, do you think it is a good method to solve the problem? Thank you.
Great!
Will the data lose the "reality" after the correction? I'm just afraid that.
Shouguang Huang Hi, just wondering if you ever solved this problem. I need to correct for photobleaching on an image stack for intensity quantification as well.
Deblurring and sharpening algorithms are helpful to restore high quality images and video. If necessary, I can give you the codes.
Justin Joseph It will be great if you can give me your codes. I am going to test them.
Sure dear Shouguang. Can you share me one set of data. [email protected] or [email protected]
I may customize the code and will give you.
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