How many photons are needed for FRET imaging?

Abstract

Förster resonance energy transfer (FRET) imaging is an essential analytical method in biomedical research. The limited photon-budget experimentally available, 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 analysis of photon-statistics in FRET imaging demonstrating how the precision of FRET imaging varies vastly with imaging parameters. Therefore, we provide analytical and numerical tools for assay optimization. Fluorescence lifetime imaging microscopy (FLIM) is a very robust technique with excellent photon-efficiencies. However, we show that also intensity-based FRET imaging can reach high precision by utilizing information from both donor and acceptor fluorophores.

Fast Timing Techniques in FLIM Applications

“…Fluorescence decay measurements in fluorescence spectroscopy or fluorescence microscopy can therefore be used to gain information about the immediate environment of the fluorophore, as well as to probe the proximity of other fluorophores via Förster Resonance Energy Transfer (FRET) [18][19][20]. Moreover, the fluorescence decay is typically independent of the fluorophore concentration at concentrations low enough to avoid interaction or aggregation.…”

Transfer Bandwidth Optimization for Multichannel Time-Correlated Single-Photon-Counting Systems Using a Router-Based Architecture: New Advancements and Results

“…In recent years, time-correlated single-photon counting (TCSPC) has become a fundamental technique for many scientific, medical, and industrial applications where it is necessary to measure fast and faint luminous signals with picosecond resolution [1]. In particular, TCSPC is applied to remote sensing, like light detection and ranging (LIDAR) [2,3], while in life science it enables high-precision analysis such as fluorescence lifetime imaging microscopy (FLIM) [4][5][6] and Förster resonance energy transfer (FRET) [7][8][9][10]. In a typical TCSPC measurement, a sample is excited by a pulsed laser source, and a histogram with the same shape as the optical curve is constructed by measuring the arrival time of each re-emitted photon [11].…”