Thanks a lot to all you people replying.
At the moment, the B-n-H time domain FLIM system is the only option
easily available for me, and it seems unlikely that our Microscope Core
Facility makes any big purchases this year. However in case a new FLIM
system is considered, I'll definitely come back to this thread to review
your kind replies and suggestions.
Other people who use the system at our institute, do live cell
measurements, as their samples are better suited for that, and they told
me that fixation decreased the lifetimes of ECFP in their constructs.
With my very preliminary data so far, I also do see a decrease in
lifetime (amplitude-weighted means of 2-exponential fit) for fixed
SCFP3A (kind gift of Dorus Gadella); within 1 day after fixation the
non-fused protein had T = 2.5 (that actually was nearly ok), but the
fused variant had T = 2.1, which in one week after fixation became 1.6.
The latter would obviously preclude any meaningful FRET measurements,
though I might still play with freshly fixed samples.
One last question I have for this thread is - what are the
recommendations for the CFP variants to use in (time-domain) FLIM: for
example, whether Cerulean is preferred over ECFP? Or do people switch
now to novel CFP variants?
Thanks again,
Best,
Alex
=============================
Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation
Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
+49 (0)30 9406 3212
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Kozlenkov, Alexey
Sent: Montag, 20. Juli 2009 14:03
To: [log in to unmask]
Subject: time-domain FLIM-FRET with fixed samples
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
=============================
Alexey Kozlenkov, PhD
Molecular Physiology of Somatic Sensation
Max-Delbruck Centrum for Molecular Medicine
13125 Berlin
Germany
+49 (0)30 9406 3212
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