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).