How many photons are needed for FRET imaging?
Biomedical optics express
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Esposito, A. (2020). How many photons are needed for FRET imaging?. Biomedical optics express, 11 (2), 1186-1202. https://doi.org/10.1364/BOE.379305
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.
External DOI: https://doi.org/10.1364/BOE.379305
This record's URL: https://www.repository.cam.ac.uk/handle/1810/304105
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/