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Date: | Fri, 30 Nov 2007 10:48:29 +0200 |
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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
>
> 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
FAX: 972-3-7384050
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