6. Fluorescence correlation spectroscopy (FCS)
FCS is a group of techniques that analyse the fluctuations in
fluorescence intensity of labelled molecules within a small observation
volume (Figure 2j). FCS techniques provide information about diffusion
times, molecular interactions, and concentration within the observed
sample with high sensitivity and temporal resolution. FCS drawbacks
include limited spatial resolution since it relies on the minute
observation volume, susceptibility to photobleaching and phototoxicity
effects, and the requirement for low concentrations of fluorescently
labelled molecules, which present a challenge for certain proteins.
FCS approaches can be broadly divided into single-point and imaging
(Figures 2 and 3, Supporting Table 1). Single-point FCS uses a fixed
detection volume and does not provide spatial resolution beyond that.
Single-point FCS can be multiplexed by simultaneously tracing multiple
molecular species (fluorescence cross-correlation spectroscopy (FCCS))
and expanded with photon counting histogram – fluorescence intensity
distribution analysis (PCH-FIDA). FCS and FCCS have been used to
characterise the ligand-binding properties of multiple GPCRs (Antoine et
al., 2016; Grime et al., 2020; Rico et al., 2019; Rose et al., 2012).
PCH-FIDA has been used in the study of mobility and oligomerisation of
several GPCRs, including serotonin, adrenergic, muscarinic, and dopamine
receptors (Herrick-Davis et al., 2013). In contrast to single-point FCS,
imaging FCS scans the sample and achieves the spatial resolution
relevant for live-cell imaging. Imaging FCS and Raster scanning
correlation spectroscopy (RICS) (Digman & Gratton, 2009) have been used
to determine the diffusion times of GPCRs and G proteins (Foust &
Piston, 2021) and the dynamics of bradykinin receptor signalling
complexes (Philip et al., 2007) in live cells. Further, comprehensive
information on single-point and imaging FCS approaches and applications
in GPCR studies can be found in (Briddon et al., 2018; Kilpatrick &
Hill, 2021).
FCS can be combined with other imaging modalities including TIRF, STED,
and light sheet fluorescence microscopy (LSFM). Total internal
reflection FCS (TIR-FCS) combines evanescent wave excitation with
fluorescence correlation spectroscopy, allowing the study of molecular
diffusion and interactions near the plasma membrane with high spatial
and temporal resolution (Lieto et al., 2003). STED-FCS combines STED
microscopy with FCS to achieve super-resolution imaging and measurement
of molecular diffusion and interactions with the nanometre spatial
resolution (Sezgin et al., 2019). It utilises a STED laser to narrow the
effective PSF, allowing for highly localised fluorescence excitation and
precise correlation analysis. Selective Plane Illumination Microscopy
FCS (SPIM-FCS) combines the advantages of LSFM and FCS techniques,
allowing for non-invasive three-dimensional measurements of molecular
diffusion and interactions in live cells or tissues with high
spatiotemporal resolution (Wohland et al., 2010). Further FCS
development, particularly in combination with SRN techniques, will
provide dynamic insights into signalling processes at very high temporal
and spatial resolution.