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Ammasi Periasamy wrote:

  > -----------------------
> For acceptor photobleaching Alexa dye groups are not good.
> Because the Alexa dyes were genrated to reduce the
> photobleaching problem in fluorescence imaging. There is
> always a problem in acceptor photobleaching either you use
> FLIM or intensity based FRET. The acceptor excitation
> always bleach the donor molecule to some extend because of
> the close spectral overlap.

We did acceptor photobleaching with a TRITC/Alexa633 D/A pair,
and got decent results. There was no measurable TRITC bleaching
with the 633nm acceptor excitation. Alexa dyes may be more stable
than the fluorescein that they were originally designed to replace, but
they still bleach.

The reason this pair was used is because the FITC/TRITC experiment
was compromised by the FITC bleaching observed with the 543nm excitation.
There are probably better pairs, but this is what we had.

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

Ph:  972-3-5317638
FAX: 972-3-5340697