7. Polarisation microscopy
The excitation probability of most existing fluorophores is sensitive to the excitation light polarisation. Therefore, fluorophore excitation and fluorescence emission when using linearly or circularly polarised light depend on molecular orientation and yield information on protein conformation and protein-protein interactions, particularly in membrane-localised systems (Figure 2k) (Lazar et al., 2011). This phenomenon has been used to determine the extent of dissociation of heterotrimeric G proteins upon activation (Bondar & Lazar, 2014) and the detection of pre-existing complexes between GPCRs and G proteins (Bondar & Lazar, 2017). Recent advances in the characterization of fluorescent proteins (Myskova et al., 2020) enabled quantitative structural insights into membrane protein conformations using modulation of the excitation light polarisation (Bondar et al., 2021; Rimoli et al., 2022). Moreover, polarisation microscopy has been combined with STORM for the superresolution imaging of dense samples (Rimoli et al., 2022). Polarisation microscopy remains an underutilised tool, likely due to the relatively complex implementation and data analysis, but it holds great promise in expanding the capabilities of other available imaging techniques. Importantly, fluorophore sensitivity to excitation light polarisation is ubiquitously present in the imaging data even if it is not specifically desired by the experiment and needs to be either accounted for or eliminated (Pulin et al., 2022).