摘要
冷冻电子断层扫描成像(cryo-ET)是细胞原位解析生物大分子结构的核心技术,cryo-ET的样品厚度需要小于300 nm,冷冻样品聚焦离子束减薄(FIB)是样品制备流程中的必要环节。当前,FIB存在难以迅速精确定位目标区域的问题,原位冷冻光电关联技术(cryo-CLEM)是一项新兴的技术,对原位冷冻样品分别进行冷冻光镜成像和电镜成像,结合了荧光成像的定位优势和电镜成像的分辨率优势,通过将光镜和电镜图像进行配准,指导FIB对原位冷冻样品减薄,能够极大地提高cryo-ET的样品制备效率。本文介绍了cryo-CLEM中的原位冷冻技术和光电关联成像技术的最新进展和应用情况,重点讨论了超分辨cryo-CLEM成像技术以及嵌入式cryo-CLEM技术,分析了各种方法的优缺点和适用范围,并对cryo-CLEM技术当前面临的主要限制和未来的发展方向进行了展望。
Cryo-electron tomography(cryo-ET) has become a cutting-edge technology in life sciences for the investigation of protein complexes directly in their natural state. In cryo-ET, the sample’s thickness must be less than 300 nm and the target molecule must be within the lamella, which is prepared by cryo-Focus Iron Beam(FIB) milling. In order to precisely navigate molecules and to improve the efficiency of sample preparation, cryo-Correlative Light and Electron Microscopy(cryo-CLEM) has been introduced to perform in-situ imaging on the frozen samples. The cryo-CLEM combines the localization advantages of fluorescence imaging with the resolution advantages of electron microscopy. By registering images of light and electrons,frozen samples can be thinned by FIB milling, so the efficiency of cryo-ET sample preparation can be improved. In this paper, we review the latest progress and applications of cryo-CLEM technologies, with a particular focus on super-resolution cryo-CLEM imaging and integrated cryo-CLEM. The advantages and limitations of various methodologies, as well as their application scope, are discussed. A discussion on cryoCLEM’s limitations and potential directions for its future development are also presented.
作者
卢婧
李尉兴
徐晓君
纪伟
LU Jing;LI Wei-xing;XU Xiao-jun;JI Wei(Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China)
出处
《中国光学（中英文）》
EI
CAS
CSCD
北大核心
2022年第6期1275-1286,共12页
Chinese Optics
基金
国家重点研发计划项目(No.2021YFA1301500)
国家自然科学基金资助项目(No.62105356)
中国科学院战略性先导科技专项(No.XDB37000000)
中国科学院科研仪器设备研制项目(No.GJJSTD20210001)。
关键词
光电关联成像
荧光导航聚焦离子束减薄
光学超分辨
correlative light and electron microscopy(CLEM)
fluorescence navigated focus ion beam(FIB)milling
super resolution
