Reducing the Detrimental Effects of Saturation Phenomena in FRET Microscopy

Although Förster resonance energy transfer (FRET) is one of the most widely used biophysical methods in biology, the effect of high excitation intensity, leading to donor and acceptor saturation, has not been addressed previously. Here, we present a formalism for the experimental determination of the FRET efficiency at high excitation intensity when saturation of both the donor and the acceptor significantly affect conventional FRET calculations. We show that the proposed methodology significantly reduces the dependence of the FRET efficiency on excitation intensity, which otherwise significantly distorts FRET calculations at high excitation intensities commonly used in experiments. The work presented here adds additional rigor to the FRET-based investigation of protein interactions and strengthens the device independence of such results.