Abstract
BackgroundThe use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.Methodology/Principal FindingsWhen we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.Conclusions/SignificanceOur results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.
Highlights
Cyan and yellow fluorescent proteins (CFP and YFP, respectively) are very frequently used in a great variety of experiments employing fluorescence microscopy, because they can be distinguished quite by appropriate filter sets
We applied the fluorescence resonance energy transfer (FRET) microscopy technique that is based on bleaching of the acceptor to cells transfected with interacting CFPand YFP-tagged proteins or a positive cyan fluorescent proteins (CFP)-YFP FRET probe
We noticed that the donor fluorescence faded much faster in the mounted, fixed samples, while it appeared more difficult to bleach the acceptor. This prompted us to determine the kinetics of photobleaching of CFP and YFP in live versus fixed cells and to investigate the influence of the fluid, in which the cells are embedded for microscopy
Summary
Cyan and yellow fluorescent proteins (CFP and YFP, respectively) are very frequently used in a great variety of experiments employing fluorescence microscopy, because they can be distinguished quite by appropriate filter sets. Conclusions/Significance: Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins.
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