The pH probe CypHer™5E is effectively quenched by FM dyes

Concurrent imaging of spectrally distinct fluorescence probes has become an important method for live-cell microscopy experiments in many biological disciplines. The technique enables the identification of a multitude of causal relationships. However, interactions between fluorescent dyes beyond an obvious overlap of their fluorescent spectra are often neglected. Here we present the effects of the well-established fluorescent dyes FM®2-10 or FM®1-43 on the recently introduced pH-dependent probe CypHer™5E. Spectrophotometry as well as live-cell fluorescence microscopy revealed that both FM dyes are effective quenchers of CypHer™5E. Control experiments indicated that this effect is reversible and not due to bleaching. We conclude that, in general, parallel measurements of both dyes are possible, with low FM dye concentrations. Nevertheless, our results implicate that special care has to be taken in such dual colour experiments especially when analysing dynamic CypHer™5E signals in live-cell microscopy.