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I do agree about FLIM detection of FRET. Neverthe less we are comparing
the photobleaching method and the multiple images method (with some
variations) introduced by Ammasi Perisamy.
All my best
Alby
On 24 giu 2004, at 18:56, Iain Johnson wrote:

> 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
>> ----------------------------------------------------------------------
>> --
>> --------------------------
>
>
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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
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--------------------------