Six-dimensional single-molecule imaging with isotropic resolution using a multi-view reflector microscope

Imaging of both the positions and orientations of single fluorophores, termed single-molecule orientation-localization microscopy, is a powerful tool for the study of biochemical processes. However, the limited photon budget associated with single-molecule fluorescence makes high-dimensional imaging with isotropic, nanoscale spatial resolution a formidable challenge. Here we realize a radially and azimuthally polarized multi-view reflector (raMVR) microscope for the imaging of the three-dimensional (3D) positions and 3D orientations of single molecules, with precisions of 10.9 nm and 2.0 & DEG; over a 1.5-mu m depth range. The raMVR microscope achieves 6D super-resolution imaging of Nile red molecules transiently bound to lipid-coated spheres, accurately resolving their spherical morphology, despite refractive-index mismatch. By observing the rotational dynamics of Nile red, raMVR images also resolve the infiltration of lipid membranes by amyloid-beta oligomers without covalent labelling. Finally, we demonstrate 6D imaging of cell membranes, where the orientations of specific fluorophores reveal heterogeneity in membrane fluidity. With its nearly isotropic 3D spatial resolution and orientation measurement precision, we expect the raMVR microscope to enable 6D imaging of molecular dynamics within biological and chemical systems with exceptional detail.

Automated summary

Single-molecule orientation-localization microscopy is a powerful tool for studying biochemical processes, but the limited photon budget poses challenges for high-dimensional imaging. A raMVR microscope has been developed with isotropic 3D spatial resolution and precision in measuring molecular orientations, enabling detailed imaging of molecular dynamics within biological and chemical systems.