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The 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 and tracing of specific proteins or cells or to determine protein interactions. In the later case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is visualized by specific microscopy techniques. When we applied a commonly used FRET microscopy technique - the increase in CFP-fluorescence after bleaching of YFP, we noticed that it worked well for live cells, but that most of the FRET-signal was lost in fixed cells mounted in commercial microscopy mounting fluids. Subsequently, we could show that CFP bleached much faster in the mounting medium than in live cells, while the opposite effect was observed for YFP. This change in photostability was not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increased in live cells after illumination at the YFP-excitation wavelength – a phenomenon, which might cause a false-positive signal in the FRET-microscopy technique that is based on bleaching of YFP. All together our results show that it is problematic to use commercially available mounting fluids for fluorescent proteins due to their differential effects on the bleaching kinetics and that the FRET microscopy technique based on bleaching of the acceptor is prone to artefacts at least for the CFP/YFP pair.

CFP and YFP photostabilities are differentially affected by common mounting fluids