The curvature of biological membranes at the nanometer scale is criticallyimportant for vesicle trafficking, organelle morphology, and diseasepropagation. This manuscript reports the development of Polarized LocalizationMicroscopy (PLM), a pointillist optical imaging technique for the detection ofnanoscale membrane curvature in correlation with single-molecule dynamics andmolecular sorting. PLM combines polarized total internal reflectionfluorescence microscopy (TIRFM) and single-molecule localization microscopy toreveal membrane orientation with sub-diffraction-limited resolution withoutreducing localization precision by point spread function (PSF) manipulation.Further, membrane curvature detection with PLM requires fewer localizationevents to detect curvature than 3D single-molecule localization microscopy(e.g., PALM or STORM), which enables curvature detection 10x faster via PLMthan via 3D single-molecule localizations. With high sensitivity, PLM detectscurvature with provides super resolution images with >10x signal-to-noiseenhancement from diffraction-limited polarized TIRFM. With rotationallyconfined lipophilic fluorophores and the polarized incident fluorescenceexcitation, membrane-bending events are revealed with super-resolution.Engineered hemispherical membrane curvature with a radius >= 24 nm was detectedwith PLM with individual fluorophore have a localization precision of 13 +/- 5nm. Further, deciphering molecular dynamics as a function of membrane topologywas enabled. The diffusion coefficient of individual DiI molecules was 7.6xhigher in planar supported lipid bilayers than within nanoscale membranecurvature.
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