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).