5. Single-particle tracking
Ensemble imaging techniques often require overexpression of the
fluorescently labelled protein of interest and cannot provide insights
into the behaviour of individual molecules and the degree of variability
between them. In contrast, SPT acquires information at the resolution of
individual molecules (Figure 2i). SPT uses diffraction of the emitted
fluorescence from a fluorescent molecule to accurately determine its
subpixel localization in the image and advanced image processing to
track its movement with a millisecond temporal resolution. SPT can work
at low expression levels that often occur in endogenous systems and
achieves the multiplexing of signals through simultaneous tracking of
different fluorophores (Sotolongo Bellon et al., 2022). The limitations
of SPT include its limited usability for molecules localised outside of
the plasma membrane, demanding sample preparation, complex data
analysis, and potential underexpression of the target protein below the
expected endogenous level. SPT is commonly performed using Total
internal reflection fluorescence (TIRF) microscopy, which achieves high
contrast by selectively exciting only fluorophores localised in the
close vicinity (typically 80-150 nm) to the coverslip surface (Axelrod,
1981). TIRF is prominently suitable for imaging molecules localised in
the plasma membrane, and therefore TIRF and SPT are widely used for the
study of GPCR signalling.
In the research of GPCR signalling components, SPT has been used to
detect their molecular mobility (Calebiro et al., 2013; Petelák et al.,
2023; Rosier et al., 2021; Sungkaworn et al., 2017), localisation
(Bondar et al., 2020; Eichel et al., 2018; Gormal et al., 2020; Grimes
et al., 2023; Sungkaworn et al., 2017), the dynamics of intermolecular
interactions (Grimes et al., 2023; Sungkaworn et al., 2017), and
di-oligomerisation (Calebiro et al., 2013; Kasai et al., 2018; Latty et
al., 2015; Moller et al., 2020). SPT has been used to uncover the modes
of interaction between GPCRs and β-arrestins and the dynamics of
accumulation of these signalling molecules in membrane domains,
particularly in clathrin-coated pits (Bondar et al., 2020; Eichel et
al., 2018; Grimes et al., 2023). SPT results have led to the concept of
“hot spots” postulating the compartmentalisation of GPCR signalling
into confined areas defined by the cytoskeleton in the plasma membrane
(Sungkaworn et al., 2017). SPT can be combined with other techniques
such as smFRET (Asher et al., 2021) and FLIM-FRET (Graham et al., 2022)
in living cells to verify detected protein-protein interactions. The
further automation of data acquisition and analysis for SPT (Yasui et
al., 2018) will improve its experimental throughput rates. Recently
developed fluorescent GPCR ligands with subnanomolar affinity compatible
with SPT (Gentzsch et al., 2020; Isbilir et al., 2020; Rosier et al.,
2021) possess a high potential for imaging endogenous GPCRs in living
cells. Overall, SPT will likely find more applications in the imaging of
conformational dynamics, component localisation, and protein-protein
interactions in endogenous signalling systems.