Hi Anke,
I am copying an abstract from a paper describing the photosenstivity of
CFP . Perhaps the problem is not the detector but the CFP itself. I have
also seen this decrease in lifetime with CFP. If you have Ceruleun it is
not as photosensitive as CFP.
Best,
Michelle

Research Article
Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on
FRET determination by fluorescence lifetime imaging microscopy in living
cells
Marc Tramier, Morad Zahid, Jean-Claude Mevel, Marie-Jo Masse, Maïté
Coppey-Moisan *


Abstract
Fluorescent protein-based FRET is a powerful method for visualizing
protein-protein interactions and biochemical reactions in living cells. It
can be difficult, however, to avoid photobleaching when observing
fluorescent cells under the microscope, especially those expressing CFP.
We compared the sensitivity of two protein-based FRET pairs to
light-induced fluorescence changes in the donor, on FRET determination by
fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low
excitation light levels of the time- and space-correlated single photon
counting (TSCSPC) method, FLIM acquisitions were achieved without donor
photobleaching. Here, we show that photobleaching of CFP by a mercury lamp
under the microscope induced a decrease in the mean fluorescence lifetime,
which interfered with FRET determination between CFP and YFP. Importantly,
the range of light-induced variation of the mean fluorescence lifetime of
CFP was not proportional to the decrease in the steady state fluorescence
intensity and varied from cell to cell. The choice of the CFP/YFP pair
therefore requires that the cells be observed and analyzed at very low
light levels during the whole FRET experiment. In contrast, the
GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if
some GFP photobleaching took place. We thus demonstrate that CFP can be an
unreliable donor for FRET determination in living cells, due to its
photosensitivity properties. We demonstrate that the GFP/mCherry pair is
better suited for FRET measurement by FLIM in living cells than the
CFP/YFP pair. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc.

-- 
Michelle Digman, Ph.D.
Director, Optical Biology Core
University of California, Irvine
McGaugh Hall 4443
(949) 824-3856 (OBC office)
(949) 282-8220 (mobile)
http://dbc.bio.uci.edu/index.html
Laboratory for Fluorescence Dynamics