DataSheet1_3D Single Molecule Super-Resolution Microscopy of Whole Nuclear Lamina.docx

Single molecule (SM) super-resolution microscopies bypass the diffraction limit of conventional optical techniques and provide excellent spatial resolutions in the tens of nanometers without overly complex microscope hardware. SM imaging using optical astigmatism is an efficient strategy for visualizing subcellular features in 3D with a z-range of up to ∼1 µm per acquisition. This approach however, places high demands on fluorophore brightness and photoswitching resilience meaning that imaging entire cell volumes in 3D using SM super-resolution remains challenging. Here we employ SM astigmatism together with multiplane acquisition to visualize the whole nuclear lamina of COS-7 and T cells in 3D. Nuclear lamina provides structural support to the nuclear envelope and participates in vital nuclear functions including internuclear transport, chromatin organization and gene regulation. Its position at the periphery of the nucleus provides a visible reference of the nuclear boundary and can be used to quantify the spatial distribution of intranuclear components such as histone modifications and transcription factors. We found Alexa Fluor 647, a popular photoswitchable fluorophore, remained viable for over an hour of continuous high laser power exposure, and provided sufficient brightness detectable up to 8 µm deep into a cell, allowing us to capture the entire nuclear lamina in 3D. Our approach provides sufficient super-resolution detail of nuclear lamina morphology to enable quantification of overall nuclear dimensions and local membrane features.

单分子（Single Molecule, SM）超分辨显微技术可突破传统光学技术的衍射极限，无需过于复杂的显微镜硬件即可实现数十纳米级的优异空间分辨率。采用光学像散（optical astigmatism）的单分子成像是一种高效策略，可在三维层面可视化亚细胞结构，单次采集的轴向（z轴）成像范围可达约1微米。然而该方法对荧光团（fluorophore）的亮度与光切换稳定性（photoswitching resilience）要求极高，这使得利用单分子超分辨技术实现全细胞体积的三维成像仍颇具挑战。本研究将单分子像散技术与多平面采集（multiplane acquisition）相结合，实现了COS-7细胞与T细胞全核纤层（nuclear lamina）的三维可视化。核纤层为核被膜（nuclear envelope）提供结构支撑，并参与核内诸多重要生命活动，包括核间运输（internuclear transport）、染色质组织（chromatin organization）与基因调控（gene regulation）。它定位于细胞核外周，可作为核边界的可视化参照，用于量化组蛋白修饰（histone modifications）、转录因子（transcription factors）等核内组分的空间分布。本研究发现，常用的光可切换荧光团Alexa Fluor 647可在持续高功率激光照射下保持活性超过一小时，且其亮度足以在细胞深处8微米处被检测到，从而实现了全核纤层的三维成像。本方法可获取核纤层形态足够精细的超分辨细节，从而实现细胞核整体尺寸与局部膜结构特征的定量分析。