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).