Re: FLIM-FRET

Posted by David Stanek-3 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FLIM-FRET-tp591029p591030.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I had the exact same problem and was asking people around. Finally one physicist explained that to me. Here is as I  (as a biologist) got his explanation. 

Yes, you are right that you are counting only photons from molecules that are not FRETing. BUT: Number of emitted photons in any given time depends on number of fluorochromes in an excited state: 

n=k.Ca 

where n - number of photons; 

k - probability that they leave the excited state;

Ca - number of activated molecules .  

In "NO FRET" situation excited molecules can relax only (forget about bleaching and other stuff) through emission of a photon. But when their are FRETing they have two ways how to relax - either emit a photon or give energy to the FRET partner. That results in faster depletion of molecules in excited state which we can measure as "faster" decay. As I understand it, you actually don't change k (which has halftime hidden somewhere inside) but Ca, which as a result looks like you have faster decay. 

Hope it helps

David

David Stanek, PhD.

Department of RNA biology

Institute of Molecular Genetics AS CR

Videnska 1083

142 20 Prague 4

Czech Republic

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web: www.img.cas.cz

On Nov 26, 2007, at 10:24 AM, Kevin Braeckmans wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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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