Gao Hao-Cheng, Xu Fan, Cheng Xi, Bi Cheng, Zheng Yue, Li Yilun, Chen Tailong, Li Yumian, Chubykin Alexander A, Huang Fang
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA.
School of Optics and Photonics, Beijing Institute of Technology, Beijing 100081, China.
bioRxiv. 2025 Feb 6:2025.02.03.636258. doi: 10.1101/2025.02.03.636258.
Single-molecule super-resolution microscopy allows pin-pointing individual molecular positions in cells with nanometer precision. However, achieving molecular resolution through tissues is often difficult because of optical scattering and aberrations. We introduced 4Pi single-molecule nanoscopy for brain with point spread function retrieval through opaque tissue (4Pi-BRAINSPOT), integrating 4Pi single-molecule switching nanoscopy with dynamic coherent PSF modeling, single-molecule compatible tissue clearing, light-sheet illumination, and a novel quantitative analysis pipeline utilizing the highly accurate 3D molecular coordinates. This approach enables the quantification of protein distribution with sub-15-nm resolution in all three dimensions in complex tissue specimens. We demonstrated 4Pi-BRAINSPOT's capacities in revealing the molecular arrangements in various sub-cellular organelles and resolved the membrane morphology of individual dendritic spines through 50-μm transgenic mouse brain slices. This ultra-high-resolution approach allows us to decipher nanoscale organelle architecture and molecular distribution in both isolated cells and native tissue environments with precision down to a few nanometers.
单分子超分辨率显微镜能够以纳米精度精准定位细胞内单个分子的位置。然而，由于光学散射和像差，在组织中实现分子分辨率往往很困难。我们引入了用于脑成像的4Pi单分子纳米显微镜技术，即通过不透明组织进行点扩散函数检索的4Pi-BRAINSPOT，它将4Pi单分子切换纳米显微镜与动态相干点扩散函数建模、单分子兼容的组织透明化处理、光片照明以及利用高精度三维分子坐标的新型定量分析流程相结合。这种方法能够在复杂组织标本的所有三个维度上以低于15纳米的分辨率对蛋白质分布进行定量分析。我们展示了4Pi-BRAINSPOT在揭示各种亚细胞器中分子排列方面的能力，并通过50微米厚的转基因小鼠脑切片解析了单个树突棘的膜形态。这种超高分辨率方法使我们能够在分离细胞和天然组织环境中精确到几纳米的精度来解读纳米级细胞器结构和分子分布。
