Re: FLIM-FRET

Posted by Aryeh Weiss on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FLIM-FRET-tp591029p591035.html

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Kevin Braeckmans wrote:

> 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.
>

In the event of 100% FRET efficiency (I should only be so lucky...) you might
detect it due to a change in acceptor lifetime. The acceptor emission will be
non-exponential, but on average, FRET excited acceptors should have longer
emission times than directly excited acceptors.

As for acceptor photobleaching, 100% efficiency would be great, because the
donor will be zero prebleach, and will go up to whatever postbleach. The limit
will be how precisely you can measure the zero.

--aryeh
--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
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