3.2 PALM and STORM
PALM and STORM techniques published just one day apart (Betzig et al., 2006; Rust et al., 2006), often referred to as single-molecule localization microscopy (SMLM) or “blinking techniques”, share the same principle of selectively exciting a small number of fluorescent molecules in the sample (Figure 2b). PALM relies on the photoswitching of fluorescent proteins (FPs), while STORM relies on the blinking of organic dyes. The precise localisation of spatially separated molecules by these techniques is determined by the deconvolution of a point spread function (PSF) from the raw imaging data. This allows the achievement of a resolution of a few tens of nanometres. PALM and STORM have been used to study the nanoscale organisation of GPCR signalling components in cellular compartments and GPCR oligomerisation (Dudok et al., 2015; Jonas et al., 2015; Moller et al., 2020; Scarselli et al., 2012; Siddig et al., 2020). SMLM techniques allowed the detection of cytoskeleton-dependent β2-adrenoceptor (β2AR) clusters (Scarselli et al., 2012), and the accurate localisation of metabotropic glutamate receptors (mGluRs) (Siddig et al., 2020) and cannabinoid receptor type 1 (Dudok et al., 2015) in synaptic zones. SMLM has also been used to detect and quantify GPCR oligomers with PALM (Jonas et al., 2015) and direct STORM allowing the determination of the exact oligomer composition (Moller et al., 2020). An interesting application of PALM combined with SPT allowed imaging of GPCR mobility with single-molecule precision in a dense environment typically inaccessible for SPT measurements (Eichel et al., 2018). Conveniently, SMLM probes can be attached not only to proteins of interest but also to small molecules, such as GPCR ligands, as has been shown for the corticotropin-releasing hormone type 1 receptor (Szalai et al., 2018), which makes these techniques very versatile and suitable for the study of endogenous systems. The main limitations of SMLM include slow data acquisition rates, localisation artefacts, large amounts of required data for processing, and time-consuming analysis. However, SMLM can be combined with photoswitching fingerprint analysis or polarisation microscopy to bypass some of these limitations.