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

Abstract

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.

Deep-SMOLM: Deep Learning Resolves the 3D Orientations and 2D Positions of Overlapping Single Molecules with Optimal Nanoscale Resolution

“…Experimental 5D imaging of amyloid fibrils Amyloid aggregates are linked to various pathological diseases, e.g., Parkinson's and Alzheimer's disease [44,45]. Previous studies have shown that upon binding transiently to amyloid fibrils, Nile red (NR) orients itself parallel to their backbones [3,4,15]. Thus, imaging NR enables us to validate Deep-SMOLM's performance for 5D SM imaging of orientations and positions from experimental data.…”

“…Researchers have used molecular orientations [1] to elucidate the architectures of amyloid aggregates [2][3][4], the organization of proteins in actin filaments [5,6], and changes in lipid membrane polarity and fluidity induced by cholesterol [4,7,8]. To effectively use limited photon budgets in single-molecule (SM) imaging, dipole-spread functions (DSFs), i.e., the vectorial extension of the optical microscope's point-spread function, must be engineered to encode additional information about a molecule's 3D orientation [5,[9][10][11][12][13][14][15]. However, simultaneously estimating the 3D orientation and position of an emitter is challenging because 1) it is difficult to estimate SM parameters in 5-dimensional space (3D orientation, wobble, 2D position) without getting trapped in local minima instigated by severe Poisson shot noise [4,14],…”

偏振荧光显微成像技术及研究进展（特邀）

“…40% 胆固醇的 DPPC SLBs 瞬间结合成像， 根据测得的角度对线进行定向和颜色编码 Fig. 8 Concept of the raMVR SMOLM [43] . [20] ; (b) comparison of diffractionlimited fluorescence images with Richardson -Lucy deconvolution results and SPoD images; (c) schematic diagram of P -TIRF [22] ; (d) P -TIRF imaging, Ring TIRF imaging, curve fitting of fixed microtubules, and comparison of Boulanger method and P -TIRF reconstruction depth map…”

“…Fig.8Concept of the raMVR SMOLM[43] . (a) Schematic of the raMVR microscope; (b) the raMVR pyramidal mirrors; (c) NR molecules transiently binding to DPPC SLBs containing 40% cholesterol surrounding a silica sphere, lines are oriented and colourcoded according to the measured polar angle…”