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.