Assessment of gate width size on lifetime-based Förster Resonance Energy Transfer parameter estimation

Förster Resonance Energy Transfer (FRET) is an optical imaging technique used in the investigation of protein interactions. FRET imaging can be executed via estimating the fluorescence lifetime of the donor molecule. For wide-field applications, gated detection methods are preferred for efficiency. However, lifetime quantitative estimation can be affected by the temporal gate width size employed in such a detection scheme. Herein, we investigate the impact of different gate widths on the estimation accuracy of specific FRET parameters. FRET parameters were estimated from in vivo experiments, with all of the characteristics of the experiments kept constant except for the gate width, which was increased from 300ps to 1,000ps in 100ps intervals. Simulations were then performed in order to determine the effect of gate width on the accuracy of parameter estimation.