Effects of tissue autofluorescence on FRET efficiency estimates

F¨orster resonance energy transfer (FRET) is a tool used for studying various biological process as well as for measuring molecular distances. This process can occur when the emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor, and the fluorophores are in close enough distance for the energy to pass from donor to acceptor non-radiatively. The efficiency of this energy transfer is dependent on the distance and orientation of the fluorophores, in addition to their overlapping spectra. Here we present a study to assess the impact of tissue autofluorescence on estimates of FRET efficiency and fluorophore abundance within experimental cellular images in tissue. To accomplish this we performed a theoretical sensitivity analysis on FRET rat kidney control images with varying ranges of donor and acceptor fluorophores to observe their pixel by pixel responses. In the experimental data, the donor was the Turquoise fluorescent protein and the acceptor fluorophore was the Venus fluorescent protein. Detection of the acceptor was more difficult due to its excitation spectrum closely resembling the autofluorescence spectrum from the base image while the emission spectrum of the Turquoise donor was more unique and easier to detect. In addition, variable FRET efficiencies were added to data at different fluorophore levels to compare the visible abundance of FRET to the autofluorescence in the resultant images. This analysis can benefit future work by furthering the understanding of the donor and acceptor concentrations needed for strong FRET measurements.