  The curvature of biological membranes at the nanometer scale is critically
important for vesicle trafficking, organelle morphology, and disease
propagation. This manuscript reports the development of Polarized Localization
Microscopy (PLM), a pointillist optical imaging technique for the detection of
nanoscale membrane curvature in correlation with single-molecule dynamics and
molecular sorting. PLM combines polarized total internal reflection
fluorescence microscopy (TIRFM) and single-molecule localization microscopy to
reveal membrane orientation with sub-diffraction-limited resolution without
reducing localization precision by point spread function (PSF) manipulation.
Further, membrane curvature detection with PLM requires fewer localization
events to detect curvature than 3D single-molecule localization microscopy
(e.g., PALM or STORM), which enables curvature detection 10x faster via PLM
than via 3D single-molecule localizations. With high sensitivity, PLM detects
curvature with provides super resolution images with >10x signal-to-noise
enhancement from diffraction-limited polarized TIRFM. With rotationally
confined lipophilic fluorophores and the polarized incident fluorescence
excitation, membrane-bending events are revealed with super-resolution.
Engineered hemispherical membrane curvature with a radius >= 24 nm was detected
with PLM with individual fluorophore have a localization precision of 13 +/- 5
nm. Further, deciphering molecular dynamics as a function of membrane topology
was enabled. The diffusion coefficient of individual DiI molecules was 7.6x
higher in planar supported lipid bilayers than within nanoscale membrane
curvature.
