Various super-resolution imaging techniques have been developed to break the diffraction-limited resolution of light microscopy. However, it still remains challenging to obtain three-dimensional (3D) super-resolution information of structures and dynamic processes in live cells at high speed. We recently developed high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy and its two-dimensional (2D)-to-3D transformation algorithm to provide an effective approach to achieving 3D sub-diffraction-limit information in subcellular structures and organelles that have rotational symmetry. In contrast to most other 3D super-resolution microscopy or 3D particle-tracking microscopy approaches, SPEED microscopy does not depend on complex optical components and can be implemented onto a standard inverted epifluorescence microscope. SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside sub-micrometer biological channels or cavities at high spatiotemporal resolution. After data collection, post-localization 2D-to-3D transformation is applied to obtain 3D super-resolution structural and dynamic information. The complete protocol, including cell culture and sample preparation (6–7 d), SPEED imaging (4–5 h), data analysis and validation through simulation (5–13 h), takes ~9 d to complete.

已经开发了各种超分辨率成像技术来打破光学显微镜的衍射极限分辨率。然而，高速获取活细胞结构和动态过程的三维（3D）超分辨率信息仍然具有挑战性。我们最近开发了高速单点边缘激发亚衍射 (SPEED) 显微镜及其二维 (2D) 到 3D 转换算法，以提供一种在亚细胞结构中实现 3D 亚衍射极限信息的有效方法和具有旋转对称性的细胞器。与大多数其他 3D 超分辨率显微镜或 3D 粒子跟踪显微镜方法相比，SPEED 显微镜不依赖于复杂的光学组件，并且可以在标准倒置落射荧光显微镜上实施。SPEED 显微镜专门设计用于以高时空分辨率获得亚微米生物通道或腔内单个不动或移动荧光分子的 2D 空间位置。数据采集​​后，应用定位后的 2D 到 3D 转换，以获得 3D 超分辨率结构和动态信息。完整的方案，包括细胞培养和样品制备（6-7 天）、SPEED 成像（4-5 小时）、数据分析和模拟验证（5-13 小时），大约需要 9 天才能完成。应用后定位 2D 到 3D 转换来获得 3D 超分辨率结构和动态信息。完整的方案，包括细胞培养和样品制备（6-7 天）、SPEED 成像（4-5 小时）、数据分析和模拟验证（5-13 小时），大约需要 9 天才能完成。应用后定位 2D 到 3D 转换来获得 3D 超分辨率结构和动态信息。完整的方案，包括细胞培养和样品制备（6-7 天）、SPEED 成像（4-5 小时）、数据分析和模拟验证（5-13 小时），大约需要 9 天才能完成。