An Sha, Ziegler Karl Ferdinand, Zhang Peiyi, Wang Yu, Kwok Tim, Xu Fan, Bi Cheng, Matosevic Sandro, Yin Peng, Li Tongcang, Huang Fang
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
Department of Physics and Astronomy, Purdue University, West Lafayette, IN 47907, USA.
Biomed Opt Express. 2019 Dec 23;11(1):461-479. doi: 10.1364/BOE.377890. eCollection 2020 Jan 1.
Fluorescence nanoscopy has become an indispensable tool for studying organelle structures, protein dynamics, and interactions in biological sciences. Single-molecule localization microscopy can now routinely achieve 10-50 nm resolution through fluorescently labeled specimens in lateral optical sections. However, visualizing structures organized along the axial direction demands scanning and imaging each of the lateral imaging planes with fine intervals throughout the whole cell. This iterative process suffers from photobleaching of tagged probes, is susceptible to alignment artifacts and also limits the imaging speed. Here, we focused on the axial plane super-resolution imaging which integrated the single-objective light-sheet illumination and axial plane optical imaging with single-molecule localization technique to resolve nanoscale cellular architectures along the axial (or depth) dimension without scanning. We demonstrated that this method is compatible with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) and exchange-PAINT by virtue of its light-sheet illumination, allowing multiplexed super-resolution imaging throughout the depth of whole cells. We further demonstrated this proposed system by resolving the axial distributions of intracellular organelles such as microtubules, mitochondria, and nuclear pore complexes in both COS-7 cells and glioblastoma patient-derived tumor cells.
荧光纳米显微镜已成为生物科学中研究细胞器结构、蛋白质动力学及相互作用不可或缺的工具。单分子定位显微镜如今可通过横向光学切片中荧光标记的样本常规实现10 - 50纳米的分辨率。然而，要可视化沿轴向排列的结构，需要在整个细胞内以精细间隔对每个横向成像平面进行扫描和成像。这个迭代过程存在标记探针的光漂白问题，易受对准伪影影响，还限制了成像速度。在此，我们聚焦于轴向平面超分辨率成像，它将单物镜光片照明和轴向平面光学成像与单分子定位技术相结合，无需扫描即可沿轴向（或深度）维度解析纳米级细胞结构。我们证明，由于其光片照明，该方法与用于纳米级形貌成像的DNA点积累（DNA-PAINT）和交换PAINT兼容，可在整个细胞深度进行多重超分辨率成像。我们通过解析COS-7细胞和胶质母细胞瘤患者来源的肿瘤细胞中微管、线粒体和核孔复合体等细胞内细胞器的轴向分布，进一步展示了该系统。
