4.2 Time-resolved FRET (TR-FRET)
TR-FRET is a spectroscopy technique that combines the detection of energy transfer with the measurement of fluorescence emission lifetimes (Ergin et al., 2016). It removes background noise from other components of the system and enables performing ratiometric measurements. TR-FRET is often used with lanthanides that exhibit large Stokes shifts, long fluorescence lifetimes, and allow for efficient multiplexing. Recently developed lanthanide complexes (CoraFluors) have high synthetic accessibility and compatibility with most existing tracers (Payne et al., 2021). TR-FRET is most often used to characterise binding events and determine binding affinities (Raich et al., 2021). In studies of GPCR signalling, TR-FRET has been applied to detect vasopressin, oxytocin, and dopamine receptor oligomers in native tissues (Albizu et al., 2010) and characterise GPCR hetero-oligomers using ligands specifically binding to such complexes (Heuninck et al., 2019). In combination with smFRET and lanthanide RET, TR-FRET can be applied to detect conformational changes in GPCRs, particularly class C, such as mGluRs (Lecat-Guillet et al., 2023).