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.