Re: FLIM-FRET

Posted by Kevin Braeckmans on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FLIM-FRET-tp591029p591034.html

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

Dear Aryeh,

That makes sense, thank you.

That also provides me with an answer to my second original question: with
increasing FRET efficiency, the precision of the FLIM measurements will
decrease since less and less photons coming from the FRETting donor
molecules will be available (for a fixed observation time). It also follows
that FLIM cannot measure (the theoretical case of) 100% FRET efficiency.

Thanks to all who have responded. As always, the combined knowledge of this
list proves to be an invaluable resource.

Kind regards,

Kevin


Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: [phone]
Fax: [phone]

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens Aryeh Weiss
> Verzonden: donderdag 29 november 2007 16:34
> Aan: [hidden email]
> Onderwerp: Re: FLIM-FRET
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Kevin Braeckmans wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Thank you Mario and David for the clarifications. I am probably
> > overlooking something very trivial, but it is still not clear to me.
> >
> >
> >
> > I totally understand that the decay of the total number of excited
> > molecules will be faster if there is an additional decaying pathway,
> > such as FRET. My question/problem, however, is how this is measured
> in a
> > FLIM experiment? As far as I understand, the photon signal that is
> > measured is in fact coming from that subpopulation of excited donor
> > molecules which are not FRETting. So in my simple understanding I
> would
> > say that it is till k_f, i.e. relaxation through the fluorescence
> decay
> > pathway, which is selectively being measured and not the decay rate
> of
> > the entire population of excited molecules.
> >
> >
>
> Think of it this way -- that subpopulation that is not FRETting does
> not consist
> of a fixed population of molecules. Rather -- it consists of those
> molecules
> that managed to emit their photon before FRET could "steal" the energy
> from
> them. So what you see are those potential donors that emit quickly --
> the others
> have lost their energy to the acceptors and will never be seen in the
> donor channel.
>
> --aryeh
> --
> Aryeh Weiss
> School of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph:  972-3-5317638
> FAX: [phone]