The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy
The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy
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DOI：
10.1016/j.bpj.2017.07.034
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发表时间：
2017-10-17
影响因子：
3.4
通讯作者：
Kelly, Christopher V.
中科院分区：
文献类型：
--
作者：
Kabbani, Abir M.;Kelly, Christopher V.
纳米尺度下生物膜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光学显微镜方法无法分辨的长度尺度上。在这里，我们报告的发展偏振定位显微镜（PLM），点彩画光学成像技术检测纳米级膜曲率与单分子动力学和分子分选。PLM结合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取向，而不降低点扩散函数操作的定位精度。与三维单分子定位显微镜相比，使用PLM的膜曲率检测需要更少的定位事件来检测曲率（例如，光激活定位显微镜或随机光学重建显微镜），其使得曲率检测通过PLM快10倍。与旋转限制的亲脂性荧光团和偏振入射荧光激发，膜弯曲事件揭示与超分辨率。用PLM检测半径>= 24 nm的工程化半球形膜曲率，并且单个荧光团定位精度为13 +/-5 nm。此外，能够破译作为膜拓扑结构的函数的分子迁移率。单个Dil分子的扩散系数在平面支撑的脂质双层中比在纳米级膜曲率内高25 +/-5倍。通过这里提供的理论基础和实验演示，PLM有望成为一个强大的技术，揭示膜弯曲的生理长度尺度的生物物理机制。
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 10x 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 Dil molecules was 25 +/- 5x 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.