Kabbani Abir M, Kelly Christopher V
Department of Physics and Astronomy, Wayne State University, Detroit, Michigan.
Department of Physics and Astronomy, Wayne State University, Detroit, Michigan.
Biophys J. 2017 Oct 17;113(8):1782-1794. doi: 10.1016/j.bpj.2017.07.034.
The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report 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 and single-molecule localization microscopy to reveal membrane orientation with subdiffraction-limited resolution without reducing localization precision by point spread function manipulation. Membrane curvature detection with PLM requires fewer localization events to detect curvature than three-dimensional single-molecule localization microscopy (e.g., photoactivated localization microscopy or stochastic optical reconstruction microscopy), which enables curvature detection 10× faster via PLM. With rotationally confined lipophilic fluorophores and the polarized incident fluorescence excitation, membrane-bending events are revealed with superresolution. Engineered hemispherical membrane curvature with a radius ≥24 nm was detected with PLM, and individual fluorophore localization precision was 13 ± 5 nm. Further, deciphering molecular mobility as a function of membrane topology was enabled. The diffusion coefficient of individual DiI molecules was 25 ± 5× higher in planar supported lipid bilayers than within nanoscale membrane curvature. Through the theoretical foundation and experimental demonstration provided here, PLM is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales.
生物膜在纳米尺度的曲率对于囊泡运输、细胞器形态和疾病传播至关重要。膜弯曲的起始发生在大多数超分辨率光学显微镜方法无法分辨的长度尺度上。在此，我们报告了偏振定位显微镜（PLM）的发展，这是一种点彩派光学成像技术，用于检测与单分子动力学和分子分选相关的纳米级膜曲率。PLM结合了偏振全内反射荧光显微镜和单分子定位显微镜，以亚衍射极限分辨率揭示膜的取向，而不会通过点扩散函数操作降低定位精度。与三维单分子定位显微镜（如光激活定位显微镜或随机光学重建显微镜）相比，用PLM检测膜曲率所需的定位事件更少，这使得通过PLM检测曲率的速度快10倍。通过旋转受限的亲脂性荧光团和偏振入射荧光激发，以超分辨率揭示了膜弯曲事件。用PLM检测到半径≥24 nm的工程化半球形膜曲率，单个荧光团的定位精度为13±5 nm。此外，还能够将分子迁移率解读为膜拓扑结构的函数。单个DiI分子在平面支撑脂质双层中的扩散系数比在纳米级膜曲率内高25±5倍。通过本文提供的理论基础和实验证明，PLM有望成为一种强大的技术，用于揭示生理长度尺度下膜弯曲的潜在生物物理机制。
