Jacques Paysan wrote:
>> Wolfgang's reply raises an interesting and very important question which
> could be discussed here: Which influence has FRET-induced reduction of
> donor fluorescence on the results of double labeling immunofluorescence
> microscopy in general? Could it be, that other dye systems as well (such as
> FITC/TMR etc.) often don't show us the real colocalizers - due to quenching?
> Has anybody ever systematically investigated this problem - i.e. by
> comparing the results of green/red vs. blue/red dye pairs?
Rather than considering fluorescence resonance energy transfer a problem
in
colocalization, one should consider it a major advantage - if not the
ultimate
proof - in colocalization. It provides a spatial resolution not provided
by any
other optical technique. There are several strategies for testing the
existence
of quenching due to resonance energy transfer. The one most easy to
implement
is acceptor bleaching. In the cases mentioned in previous emails this
involves
bleaching the red-emitting dye. To get an impression of these technique,
you
could check articles authored or co-authored by T. M. Jovin.
--
Rik van Gorsel
Max Planck Institute for Biophysical Chemistry
Department of Molecular Biology
Am Fassberg 11
D-37077 Goettingen
Germany
e-mail: rvgorsel at mpc186.mpibpc.gwdg.de
phone: +49-551-201.1388 (office)
+49-551-201.1386 (lab)
fax: +49-551-201.1467
---
