Photon-statistics in sensitized emission FRET and FLIM: a comparative theoretical analysis

Abstract

Förster resonance energy transfer (FRET) imaging is an essential analytical method in biomedical research. Sensitized emission FRET (seFRET) and fluorescence lifetime imaging microscopy (FLIM) are the most common techniques used for the quantification of FRET. The limited photon-budget available in a typical experiment, however, imposes compromises between spatiotemporal and biochemical resolutions, photodamage and phototoxicity. The study of photon-statistics in biochemical imaging is thus important in guiding the efficient design of instrumentation and assays. Here, we show a comparative theoretical analysis of photon-statistics and provide computational tools to investigate the performance of FRET imaging by FLIM and seFRET. We show how the performance of FRET imaging can vary vastly with imaging parameters that should be thus always optimized. Therefore, we provide analytical tools, an open source computational framework and suggestions on assay optimization. FLIM is a very robust technique and provides excellent photon-efficiencies, but we show that also seFRET utilizes the available photon-budgets rather efficiently by utilizing information from both donor and acceptor fluorescence. We, also because of the utilization of the entire photon