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.