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

We report a radially and azimuthally polarized multi-view reflector (raMVR) microscope for precise imaging of the 3D positions and 3D orientations of single molecules (SMs, 10.9 nm and 2.0° precisions using 5000 photons). These precisions are ∼1.5 times better than those of existing methods for SM orientation-localization microscopy. The raMVR microscope achieves 6D super-resolution imaging of Nile red (NR) molecules transiently bound to 150 nm, 350 nm, and 1 µm-diameter lipid-coated spheres, accurately resolving their spherical morphology despite refractive-index mismatch. Simply by observing the rotational dynamics o raMVR images also resolve the infiltration of lipid membranes by amyloid-beta oligomers without covalent labeling. Finally, we demonstrate 6D imaging of HEK-293T cell membranes, where the orientations of merocyanine 540 molecules reveal heterogeneities in membrane fluidity. With its ∼2 µm depth range, nearly isotropic 3D spatial resolution, and superior orientation measurement precision, we expect the raMVR microscope to enable 6D imaging of molecular dynamics within biological and chemical systems with unprecedented detail.