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This has to be the liveliest thread we've had for some time.
Right now I'm in Portugal teaching in the Advanced Light Microscopy
in Living Cells course at the IGC in Oeiras.  These matters have
come up in discussion several times in the course.  But since Portugal
is currently playing England in the European Cup (some game called
soccer) nobody is paying any interest to this right now.

I would have thought that photobleaching would remove oxygen (triplet/
triplet interactions) so I don't really understand Iain's comment.

But my real point is that in the absence of lifetime imaging (which
I agree with all concerned to be a very good approach) why is
DONOR photobleaching not used as a critical measurement for FRET?
The point is that the presence of FRET axiomatially provides a
measure of protection against photobleaching.  So by ratioing
total fluorescence before and after a mild bleach one should get
a measure of the proportion of donor molecules involved in FRET.
Every other parameter should cancel out ...

                                                         Guy


Quoting Iain Johnson <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> This doesn't surprise me at all.  The acceptor photobleaching technique
> is not necessarily selective for the acceptor.  Excitation of the
> acceptor photosensitizes generation of singlet oxygen that then reacts
> quite indiscriminately with dyes, proteins, nucleic acids or any other
> reactive target in its path. Since the diffusive range of singlet oxygen
> in living cells is about 50 nm and therefore of the same order as the
> donor-acceptor proximity required for FRET, it is quite possible for
> singlet oxygen generated by excitation of the acceptor to bleach the
> donor.  This is one reason why FLIM detection of FRET, which does not
> suffer from this vunerability, is a superior technique to "acceptor"
> photobleaching.
>
>
> Alberto Diaspro wrote:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Dear friends,
> > in order to check some possible FRET between FITC and ALEXA 555, when
> > using bleaching of acceptor as method, we found that 543 nm bleached
> > FITC as well.
> > Did you ever experienced such a behaviour?
> > Alby
> >
> >
> > ------------------------------------------------------------------------
> > ---------------------------
> > Alberto Diaspro, Department of Physics, University of Genoa
> > Via Dodecaneso 33, 16146 Genova, Italy
> > facsimile +39-010314218 - voice +39-0103536426/480/309
> > URL: http://www.lambs.it
> > http://www.wiley.com/WileyCDA/WileyTitle/productCd-0471409200.html
> > ------------------------------------------------------------------------
> > --------------------------
>


--
Associate Professor Guy Cox
Electron Microscope Unit, F09
University of Sydney NSW 2006
+61 2 9351 3176