FLIM-FRET

Posted by Kevin Braeckmans on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FLIM-FRET-tp591029.html

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Dear fellow microscopists,

 

I would like to ask a question about FLIM-FRET. Having no practical experience with this technique, I was reading some reviews on this topic where it is explained that the FRET efficiency is deduced from a reduction in the lifetime of the donor fluorophore. I just want to make sure that I understand correctly what is exactly being measured here.

 

Let’s say we have a donor-acceptor pair having 50% FRET efficiency. This means that on multiple excitation events, there will be FRET in 50% of the cases, and 50% relaxation of the donor through other relaxation mechanisms, one of which is fluorescence decay. However, when FRET occurs, there is no emission of a donor fluorescence photon. So, when it is said that it is the donor lifetime which is measured in FLIM-FRET, it must be the lifetime measured from the donor fluorescence photons emitted when actually no FRET is taking place. Is that correct?

 

It this is so, it also follows that FLIM-FRET is necessarily incapable of measuring 100% FRET efficiency (because there will be no donor fluorescence photons emitted anymore). Or more generally, the FLIM-FRET accuracy will decrease with increasing FRET efficiency since less photons will be available. Is that correct?

 

Any explanation/input is much appreciated.

 

Thanks and kind regards,

 

Kevin

 

 

Kevin Braeckmans, Ph.D.

Lab. General Biochemistry & Physical Pharmacy

Ghent University

Harelbekestraat 72

9000 Ghent

Belgium

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