Dear all,


here is a question from a newbie venturing into the field of 2photon
FLIM-FRET measurements.

I'm considering using the Becker-Hickl time-domain FLIM / LSM NLO system
to measure FRET between some membrane proteins, fused to CFP and YFP.
The FLIM-based approach looked like an attractive option since it should
allow for analysing cells with not too highly expressed proteins of
interest, thus reducing the risk of obtaining FRET due only to membrane
overcrowding.
However, since my proteins are partially present in a highly motile pool
of vesicles, I intended to use fixed cell samples (as FLIM would require
some tens of seconds for one measurement).

Now, to my question: 
The Becker-Hickl TCSPC handbook by Wolfgang Becker makes a strong point
of NOT using fixed samples for FLIM-FRET, due to changes in lifetimes
and strongly double-exponential decay profiles. However, other
publications, such as a protocol in the Molecular Cloning "Bible", do
use fixed samples for FLIM-FRET. Thus, I would welcome any comments or
advice from the community about this matter. Is fixed sample FLIM-FRET
really not recommended, and if it is not true, what would be the best
methodology to use (and pitfalls to avoid). How important would be the
choice of particular fluorescent protein, fixation methods and mounting
media? Obviously, I would also be grateful for links to good reviews and
experimental publications that I might have missed.


Thanks in advance,

Alex

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Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation
Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
+49 (0)30 9406 3212