3.5 SRRF
In contrast to other SRN techniques, SRRF requires no specialised hardware or biological probes and achieves resolution enhancement to the level below 100 nm by analysing radial fluctuations of multiple (hundreds) images with a low signal-to-noise ratio (Gustafsson et al., 2016). SRRF can be implemented using regular FPs and fluorescent dyes on standard confocal, total internal reflection fluorescence (TIRF), and even widefield microscopes. Enhanced SRRF (eSRRF), a recent technique update, enables the achievement of improved resolution in 3D (Laine et al., 2022). So far, in studies of GPCR signalling, SRRF has been used primarily as a supporting technique to visualise actin filaments alongside SPT of β2AR-βArrestin-2 complexes (Grimes et al., 2023) or determine the cellular localisation of endogenous glucagon-like peptide-1 receptor using fluorescent peptide antagonists (Ast et al., 2020). Importantly, SRRF and eSRRF are available as free ImageJ plugins. These image processing tools show great potential to democratise the implementation of SRN in a variety of applications without the need for costly equipment and specialised experimental design.