《生命科学》 2025, 37(7): 854-867

冷冻电镜前沿技术与发展应用

宋　芸¹ , 左沁宇² , 蒙学明² , 李光一¹ , 魏宇宇¹ , 段佳琳¹ , 辛立辉¹ , 丛　尧^2,3,* , 孔亮亮^1,*

¹中国科学院上海高等研究院国家蛋白质科学研究(上海)设施，上海 201210 ²中国科学院分子细胞科学卓越创新中 心，上海生物化学与细胞生物学研究所，核糖核酸功能与应用全国重点实验室，中国科学院大学，上海 200031 ³国科大杭州高等研究院生命与健康科学学院，中国科学院大学，浙江省系统健康科学重点实验室，杭州 310024

摘　要：

随着冷冻电镜(Cryo-EM) 分辨率革命的到来，冷冻电镜技术已成为生物大分子结构解析的核心手段。这场技术革命的背后，是多维度技术的协同发展。新兴的冷冻制样技术，尤其是亲和载网及时间分辨率的冷冻制样技术，不仅具有整合生物复合体提纯与冷冻制样、降低气- 液界面影响的潜能，还有望捕捉生物过程的瞬时构象。冷冻聚焦离子束减薄结合荧光定位与高压冷冻技术，可实现对细胞或组织样品的精准减薄，助力揭示细胞原位中的“分子社会”及生物过程的时空调控。冷冻透射电镜硬件、控制软件及探测器的不断发展，极大推进了高通量、高分辨率、自动化的电镜数据采集。近年来，人工智能(AI) 与冷冻电镜技术的深度融合，正在推进冷冻电镜技术向着捕获生物大分子动态生物过程，揭示其原位时空调控和“分子社会关系”方向飞速发展，为揭示生命本质提供了强大的技术支撑。冷冻电镜技术不仅在基础研究中具有重要意义，在药物开发等应用领域也展现出巨大潜力。本文将围绕冷冻样品制备、冷冻透射电镜及探测器硬件、数据收集及AI 应用等方向展开阐述。

通讯作者：丛　尧 , Email:cong@sibcb.ac.cn 孔亮亮 , Email:kongliangliang@sari.ac.cn

Cryo-electron microscopy: frontiers, advances and applications

SONG Yun¹ , ZUO Qin-Yu² , MENG Xue-Ming² , LI Guang-Yi¹ , WEI Yu-Yu¹ , DUAN Jia-Lin¹ , XIN Li-Hui¹ , CONG Yao^2,3,* , KONG Liang-Liang^1,*

¹National Facility for Protein Science in Shanghai, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China ²State Key Laboratory of RNA Innovation, Science and Engineering, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China ³Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China

Abstract:

Over the past decade or so, cryo-electron microscopy (Cryo-EM) has emerged as a primary approach in the structural determination of biological macromolecules. Behind this revolution lies the concurrent development of all steps of the Cryo-EM workflow. Advances in Cryo-EM sample preparation, particularly affinity grids and time-resolved sample preparation method, not only being able to integrate the purification with vitrification, minimizing artifacts from water-air interface, but also show promise in capturing transient conformational intermediates during biological processes. The combination of cryo-focused ion beam (Cryo-FIB) milling with fluorescence-guided localization and high-pressure freezing allows for precise thinning of cellular or tissue samples, facilitating the revelation of in situ “molecular societies” and the spatiotemporal regulation of biological events. Developments in microscope hardware, control software, and detectors have enabled high-throughput, highresolution, and automated data acquisition in Cryo-EM. Meanwhile, the deep integration of artificial intelligence (AI) and Cryo-EM has propelled the field to new frontiers, enabling the visualisation of time-resolved macromolecular dynamics and the decoding of in situ spatiotemporal regulation, providing a powerful technique for revealing the essence of life. Cryo-EM also demonstrates enormous potential in applications such as drug development. Here, we focus our review on Cryo-EM sample preparation, instruments (both microscopes and detectors), data acquisition strategies, and AI applications in the field of Cryo-EM.

Communication Author：CONG Yao , Email:cong@sibcb.ac.cn KONG Liang-Liang , Email:kongliangliang@sari.ac.cn