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How many photons are needed for FRET imaging?

Published version
Peer-reviewed

Type

Article

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Authors

Esposito, Alessandro  ORCID logo  https://orcid.org/0000-0002-5051-091X

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.

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Journal Title

Biomedical optics express

Conference Name

Journal ISSN

2156-7085

Volume Title

11

Publisher