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原位冷冻光电关联技术的研究进展

卢婧 李尉兴 徐晓君 纪伟

卢婧, 李尉兴, 徐晓君, 纪伟. 原位冷冻光电关联技术的研究进展[J]. 中国光学(中英文), 2022, 15(6): 1275-1286. doi: 10.37188/CO.2022-0095
引用本文: 卢婧, 李尉兴, 徐晓君, 纪伟. 原位冷冻光电关联技术的研究进展[J]. 中国光学(中英文), 2022, 15(6): 1275-1286. doi: 10.37188/CO.2022-0095
LU Jing, LI Wei-xing, XU Xiao-jun, JI Wei. Recent development of cryo-correlated light and electron microscopy[J]. Chinese Optics, 2022, 15(6): 1275-1286. doi: 10.37188/CO.2022-0095
Citation: LU Jing, LI Wei-xing, XU Xiao-jun, JI Wei. Recent development of cryo-correlated light and electron microscopy[J]. Chinese Optics, 2022, 15(6): 1275-1286. doi: 10.37188/CO.2022-0095

原位冷冻光电关联技术的研究进展

基金项目: 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)
详细信息
    作者简介:

    卢 婧(1983—),女,江苏连云港人,博士。2005年于中国科学技术大学获得电子科学与技术专业学士学位,2011年于中国科学院光电技术研究所获得光学工程博士学位,2015—2017年在美国阿拉巴马大学伯明翰分校从事博士后工作,2018年至今,在中科院生物物理研究所从事博士后工作。主要从事冷冻光电关联成像,光学超分辨成像,荧光导航聚焦离子束减薄,自适应光学眼科成像等方面的研究。E-mail:jinglu@ibp.ac.cn

    纪 伟(1983—),男,安徽太和人,博士,研究员,博士生导师。2005年于华中科技大学获得生物医学工程学士学位, 2010年于中国科学院生物物理研究所获得博士学位。近年来一直从事生命科学仪器开发,发展原创性的显微成像技术方法。近五年以通讯作者发表Nature Methods论文两篇,先后入选“中国科学院关键技术人才”、中国电子显微镜学会“优秀青年学者奖”。研究成果入选中国生命科学十大进展、国家十三五科技创新成就展。E-mail:jiwei@ibp.ac.cn

  • 中图分类号: Q439

Recent development of cryo-correlated light and electron microscopy

Funds: Supported by National Key Research and Development Program of China (No. 2021YFA1301500); National Natural Science Foundation of China (No. 62105356); Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB37000000); Scientific Instrument Developing Project of the Chinese Academy of Sciences (No. GJJSTD20210001)
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  • 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。

     

  • 图 1  原位冷冻细胞光电关联成像示意图。(a)细胞培养;(b)荧光标记;(c)原位冷冻;(d)光镜成像;(e)FIB减薄;(f)cryo-ET成像

    Figure 1.  Schematic diagram of cryo-CLEM. (a) Cell culturing; (b) fluorescent labeling; (c) fast freezing; (d) fluorescent imaging; (e) FIB milling; (f) cryo-ET imaging

    图 2  商用原位冷冻设备。(a)FEI公司VitrobotTM;(b)Gatan公司CP3;(c)Leica公司EMPact;(d)Bal-Tec公司HPM

    Figure 2.  Commercial plunge freezers and high pressure freezers. (a) VitrobotTM from FEI; (b) CP3 from Gatan; (c) EMPact from Leica; (d) HPM from Bal-Tec

    图 3  几种商用冷台和冷台样机实物图。(a)Instec公司的CLM77K;(b)Linkam公司的CMS196;(c)FEI公司的CorrSight;(d)Li等人提出的高稳定性冷台[19];(e)徐涛组提出的高稳定冷台[20]

    Figure 3.  Commercial cryo stages and prototypes. (a) CLM77K from Instec; (b) CMS196 from Linkam; (c) CorrSight from FEI; (d) Cryo stage proposed by Li[19]; (e) Cryo stage proposed by Xu[20]

    图 4  (a)分体式cryo-CLEM的成像系统示意图;(b)分体式cryo-CLEM的成像流程

    Figure 4.  (a) Schematic diagram and (b) flow chart of imaging process of independent cryo-CLEM system

    图 5  几种超分辨冷冻荧光显微镜示意图。(a)冷冻STED成像;(b)冷冻单分子定位成像;(c)冷冻结构光照明成像;(d)冷冻Airyscan成像

    Figure 5.  Schematic diagrams of cryo supper resolution fluorescent microscopy. (a) cryo-STED; (b) cryo-SMLM; (c) cryo-SIM; (d) cryo-Airyscan

    图 6  嵌入式cryo-CLEM成像系统的(a)示意图和(b)成像流程

    Figure 6.  (a) Schematic diagram and (b) block diagram of imaging process of integrated cryo-CLEM system

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  • 收稿日期:  2022-05-10
  • 修回日期:  2022-06-14
  • 网络出版日期:  2022-10-11

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