Abstract
Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.
Highlights
Förster or fluorescence resonance energy transfer (FRET), first described by Theodor Förster in 1946, is a physical phenomenon in which a donor fluorophore in its excited state non-radiatively transfers its excitation energy to a neighboring acceptor fluorophore, thereby causing the acceptor to emit its characteristic fluorescence [1]
Since FRET is highly sensitive to the distance between donor and acceptor dipoles within the 1–10 nm range, FRET-based biosensors, composed of fluorophores and sensing domains, have been widely adopted as spectroscopic rulers to monitor a variety of biochemical activities that produce changes in molecular proximity, such as protein–protein interactions, conformational changes, intracellular ion concentrations, and enzyme activities [2,3]
Depending on whether the two fluorophores are conjoined to the same molecule, FRET biosensors can be classified into two categories: (1) intramolecular type, in which donor and acceptor fluorophores are conjoined to the same molecule, whereby conformational changes in the molecule induce FRET
Summary
Förster or fluorescence resonance energy transfer (FRET), first described by Theodor Förster in 1946, is a physical phenomenon in which a donor fluorophore in its excited state non-radiatively transfers its excitation energy to a neighboring acceptor fluorophore, thereby causing the acceptor to emit its characteristic fluorescence [1]. Two categories of measurement methods have been developed to measure the FRET change: (1) indirect, which involves measurements of FRET E at different states through spectral imaging FRET (siFRET), acceptor photobleaching FRET (apFRET), and fluorescence lifetime imaging FRET (FLIM-FRET); (2) direct, which directly relates change of fluorescence intensity and polarization to the FRET change The spectral cross-talk and direct acceptor excitation are not big issues because only donor fluorescence lifetime is measured in FLIM-FRET imaging [4]. In polarization-resolved FRET imaging (prFRET), energy transfer can be detected by monitoring changes in polarization through steady-state or time-resolved measurements in the time-domain or frequency-domain and using scanning or wide-field microscopes, in which the intensities of fluorescence polarized parallel and perpendicular to the polarization vector of the polarized excitation source are measured [28]. Three-filter cube-like corrections are required to eliminate those false positives in hetero-FRET anisotropy [31]
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