4.3 FRET with fluorescence lifetime imaging microscopy (FRET-FLIM)
FRET-FLIM is an imaging technique, similar in principle to TR-FRET, that provides spatially resolved data (Figure 2h). The lifetime of emitted fluorescence provides a well-defined characteristic feature that can be used to identify the source of detected photons and the environment in the sample (Liput et al., 2020). FRET-FLIM allows accurate quantification of FRET measurements, eliminating reliance solely on widely varying fluorescence intensities. FRET-FLIM has been applied to detect the distribution of the dopamine receptor type 2 and the Gi proteins in membrane nanodomains (Polit et al., 2020) and uncover the plasma membrane-localised homodimers of the GPR17 receptor that controls the central nervous system myelination (Yang et al., 2020). FRET-FLIM has also been used for the detection of transient signalling processes such as cAMP degradation by phosphodiesterases (Harkes et al., 2021). Importantly, FRET-FLIM is compatible with deep tissue imaging with 2P microscopy and can be used for the quantitative analysis of endogenous GPCR signalling in brain slices (Chen et al., 2014).