Six-Dimensional Single-Molecule Imaging with Isotropic Resolution using a Multi-View Reflector Microscope

Imaging both the positions and orientations of single fluorophores, termed single-molecule orientation-localisation microscopy, is a powerful tool to study 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 realise a radially and azimuthally polarized multi-view reflector (raMVR) microscope for the imaging of the 3D positions and 3D orientations of single molecules, with precision of 10.9 nm and 2.0° over a 1.5 μm depth range. The raMVR microscope achieves 6D super-resolution imaging of Nile red (NR) molecules transiently bound to lipid-coated spheres, accurately resolving their spherical morphology despite refractive-index mismatch. By observing the rotational dynamics of NR, 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.