Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching.

Fluorescence resonance energy transfer (FRET) is a technique used for the study of functional interactions between molecules. The intimate vicinity between two fluorescent molecules (FRET-pair; donor and acceptor) allows for an energy transfer, which can be directly calculated as the so called FRET efficiency. This technique is used in fixed as well as living cells. Here we show first, measured by the FRET technique, that the ICln ion channel is transposed from the cytosol towards the cellular membrane in HEK cells after swelling, and second, that the calculation of the FRET efficiency by de-quenching the donor cyan-fluorescent-protein (CFP) emission due to acceptor-photobleaching leads to erroneous estimate of the FRET efficiency in fixed, mounted and sealed specimens. The acceptor photobleaching leads to a modification of the donor cyan-fluorescent-protein, which shows then a strong emission, thus mimicking functional interaction between CFP (donor) and yellow-fluorescent-protein (YFP; acceptor). Moreover, the procedure of acceptor photobleaching masks physiological (non random) interaction between molecules within the fixed, mounted and sealed cell. We show that no artifactual CFP modifications arise when using the acceptor photobleaching technique under in vivo conditions, and we offer strategies to minimize erroneous FRET efficiency calculations if cells need to be fixed.