3.1 STED
The STED technique, originally developed in the laboratory of Stefan Hell (Hell & Wichmann, 1994), achieves improved lateral and axial resolution by depleting the fluorescence emission from all molecules in a defined area except for a tiny spot less than 50 nm in the lateral direction and less than 100 nm in the axial direction (Figure 2a). In GPCR signalling research, STED has been used to determine precise localisation and evaluate clustering of cannabinoid receptor type 1 in neurons (Li et al., 2020) and uncover the distribution of endogenous glucagon-like peptide-1 receptor molecules (Ast et al., 2020). A detailed protocol for the use of ground state depletion microscopy (GSDM), a variant of STED for GPCR studies, is described in (Caetano Crowley et al., 2019). Limitations of STED include the high laser powers required for the efficient fluorescence depletion of molecules and relatively slow image acquisition. New approaches, including MINFLUX and MINSTED, described below, allow for overcoming these limitations.