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).