4. Resonance energy transfer (RET) techniques
FRET and BRET have been used for studies of GPCR signalling for more than two decades. RET techniques are based on the non-radiative transfer of energy from an excited fluorophore (donor) to another, closely located (2-10 nm), fluorophore with lower excitation energy (acceptor) which emits fluorescence (Förster, 1948). RET can be used for the detection of molecular proximity and conformational changes within a molecule at high temporal resolution. FRET and BRET have been reviewed multiple times elsewhere (Kauk & Hoffmann, 2018; Kroning & Wang, 2022; Olsen & English, 2023). Here we describe advanced RET applications, single-molecule, time-resolved, and fluorescence lifetime imaging FRET, which allow the observation of protein conformational changes and signalling dynamics in living samples with molecular resolution and structural insights (Figure 2 and 3, Supporting Table 1).