Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used 3D nanostructures such as nanopillars to imprint well-defined membrane curvatures (the “nano-bio interface”). In these studies, proteins and their interactions were probed by 2D fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone oil immersion objective and 3D double-helix point-spread function microscopy. We carefully optimize the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

中文翻译：

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用 3D 超分辨率显微镜探索细胞表面与纳米柱的相互作用

已显示质膜形貌强烈影响许多细胞过程的行为，例如网格蛋白介导的内吞作用、肌动蛋白重排等。最近的研究使用 3D 纳米结构，如纳米柱来印记明确定义的膜曲率（“纳米生物界面”）。在这些研究中，通过二维荧光显微镜探测蛋白质及其相互作用。然而，这些方法的低分辨率和有限的轴向细节并不是确定蛋白质沿 100 nm 直径物体的相对空间位置和分布的最佳选择，该物体低于光学衍射极限。在这里，我们介绍了一种通用方法，使用 3D 单分子超分辨率 (SR) 定位显微镜以 10-20 nm 的精度探索纳米生物界面上蛋白质的纳米级分布。这是通过结合硅油浸渍物镜和 3D 双螺旋点扩散函数显微镜来实现的。我们仔细优化了目标，以最大限度地减少石英纳米柱和电池之间的球面像差。为了验证 3D SR 方法，我们对表面标记的纳米柱的 3D 形状进行了成像，并将结果与​​电子显微镜测量结果进行了比较。转向细胞中的跨膜锚定标签，高质量的 3D SR 重建显示膜紧紧包裹在纳米柱周围。有趣的是，参与网格蛋白介导的内吞作用的细胞质蛋白 AP-2 沿着纳米柱积累，超过 1/R 膜曲率的特定阈值。最后，我们观察到 AP-2 和肌动蛋白优先在柱帽附近以正高斯曲率积累。