CFP and YFP photostabilities are differentially affected by common mounting fluids To the Editor:

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 easily by appropriate filter sets. Especially the original mammalian optimized versions, also known as enhanced cyan and yellow fluorescent proteins (ECFP and EYFP) are commonly applied, as they have been among the first spectrally distinct variants of green fluorescent protein1. Moreover, they are well suited for fluorescence resonance energy transfer (FRET) microscopy to visualize protein interactions or conformational changes2. In that case, the energy of the donor fluorophore (CFP) is transferred to an acceptor fluorophore (YFP) by a dipole interaction resulting in a decrease of donor and an increase in acceptor fluorescence. This phenomenon can be visualized by various microscopy techniques. One of them is based on the increase of donor fluorescence after photobleaching of the acceptor, which eliminates the energy transfer. This is practically done by acquiring a donor (CFP) image, followed by bleaching of the acceptor (YFP) with its specific excitation wavelength and the acquisition of a second donor image3. An increase in donor fluorescence, which is usually visualized by calculating a ratio or a difference image of the two donor images, indicates close proximity of FRET donor and acceptor proteins. When we applied this technique to cells transfected with interacting CFP- and YFP-tagged proteins or a positive CFP-YFP FRET probe, we noticed a striking difference in results obtained with living cells and those generated with fixed cells after mounting them on coverslips with a commercial mounting fluid (Fig. 1A). While the donor increase was perfectly visible in living cells, we did not observe a significant increase in fixed cells. Moreover, 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. After mounting the cells in a commercia