FRET: signals hidden within the noise.

Abstract

F€ orster resonance energy transfer (FRET) is a physical phenomenon that has been of incredible utility to the scientific community, enabling the study of a wide range of nanoscale, distance-dependent measurements. FRET occurs when energy is nonradiatively transferred from an excited fluorophore (the donor fluorophore) to a second fluorophore in near proximity (the acceptor fluorophore) (1,2). Because the rate at which energy is transferred from the donor to the acceptor fluorophore depends on the intermolecular distance raised to the sixth power, FRET provides a highly sensitive measure of intermolecular spacing (2,3). The key parameter in FRET measurements is the FRET efficiency. FRET efficiency is most often described as the ratio of the number of quantized energy events (sometimes referred to as virtual photons (4)) transferred from the donor fluorophore to the acceptor fluorophore, divided by the total number of quantized energy events (photons) absorbed by the donor fluorophore (5). Using appropriate measurement and computational techniques, FRET efficiency represents a quantifiable value describing the intermolecular spacing and orientation within a sample. For fluorophores with fixed intermolecular spacing, the value of the measured FRET efficiency has been shown to be relatively constant, regardless of the measurement technique used (6). Hence, FRET reporters can be used to provide absolute quantitative data describing interand intramolecular interactions. Computing the FRET efficiency, whether in fluorescence microscopy or flow cytometry measurements, typically requires a ratiometric calculation (7). Because FRET efficiencies often range from 20 to 60% for biological reporters, the fluorescence emission from the acceptor fluorophore, when excited via energy transfer, usually provides lower signal strength than measurements of acceptor fluorophore emission resulting from direct excitation. Performing ratiometric calculations using relatively weak signals as the inputs can result in compounding error propagation (8). Thus, while FRET, as a tool, offers the ability to probe molecular distances within images or flow cytometry data, it also introduces substantial limitations in sensitivity and variance, due to low signal strength. Low signal strength may also limit spatial and temporal resolution. When performing FRET experiments, trade-offs must usually be made between signal strength (accuracy of the FRET efficiency calculation), spatial resolution, and temporal