留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

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

doi: 10.37188/CO.2022-0095
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)
More Information
  • 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

  • [1] HYLTON R K, SWULIUS M T. Challenges and triumphs in cryo-electron tomography[J]. Iscience, 2021, 24(9): 102959. doi: 10.1016/j.isci.2021.102959
    [2] MARTYNOWYCZ M W, CLABBERS M T B, UNGE J, et al. Benchmarking the ideal sample thickness in cryo-EM[J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(49): e2108884118. doi: 10.1073/pnas.2108884118
    [3] DEROSIER D J. Where in the cell is my protein?[J]. Quarterly Reviews of Biophysics, 2021, 54: e9. doi: 10.1017/S003358352100007X
    [4] BISSON C, HECKSEL C W, GILCHRIST J B, et al. . Preparing lamellae from vitreous biological samples using a dual-beam scanning electron microscope for cryo-electron tomography: 1940-087X[R]. Menlo Park: SLAC National Accelerator Lab. , 2021.
    [5] GORELICK S, BUCKLEY G, GERVINSKAS G, et al. PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy[J]. eLife, 2019, 8: e45919. doi: 10.7554/eLife.45919
    [6] ARNOLD J, MAHAMID J, LUCIC V, et al. Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy[J]. Biophysical Journal, 2016, 110(4): 860-869. doi: 10.1016/j.bpj.2015.10.053
    [7] MARION J, LE BARS R, SATIAT-JEUNEMAITRE B, et al. Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots[J]. Journal of Structural Biology, 2017, 198(3): 196-202. doi: 10.1016/j.jsb.2017.03.008
    [8] TIAN X H, DE PACE C, RUIZ‐PEREZ L, et al. A cyclometalated iridium (III) complex as a microtubule probe for correlative super‐resolution fluorescence and electron microscopy[J]. Advanced Materials, 2020, 32(39): 2003901. doi: 10.1002/adma.202003901
    [9] TUIJTEL M W, KOSTER A J, JAKOBS S, et al. Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins[J]. Scientific Reports, 2019, 9(1): 1369. doi: 10.1038/s41598-018-37728-8
    [10] KLEIN S, WIMMER B H, WINTER S L, et al. Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies[J]. Communications Biology, 2021, 4(1): 137. doi: 10.1038/s42003-020-01567-z
    [11] CARTER S D, MAGESWARAN S K, FARINO Z J, et al. Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells[J]. Journal of Structural Biology, 2018, 201(1): 15-25. doi: 10.1016/j.jsb.2017.10.009
    [12] BHARAT T A M, HOFFMANN P C, KUKULSKI W. Correlative microscopy of vitreous sections provides insights into BAR-domain organization in situ[J]. Structure, 2018, 26(6): 879-886.e3. doi: 10.1016/j.str.2018.03.015
    [13] WILFLING F, LEE C W, ERDMANN P S, et al. A selective autophagy pathway for phase-separated endocytic protein deposits[J]. Molecular Cell, 2020, 80(5): 764-778.e7. doi: 10.1016/j.molcel.2020.10.030
    [14] ZHENG ZH H, LAURITZEN J S, PERLMAN E, et al. A complete electron microscopy volume of the brain of adult Drosophila melanogaster[J]. Cell, 2018, 174(3): 730-743.e22. doi: 10.1016/j.cell.2018.06.019
    [15] LIU Y T, TAO CH L, ZHANG X K, et al. Mesophasic organization of GABAA receptors in hippocampal inhibitory synapses[J]. Nature Neuroscience, 2020, 23(12): 1589-1596. doi: 10.1038/s41593-020-00729-w
    [16] TURK M, BAUMEISTER W. The promise and the challenges of cryo‐electron tomography[J]. FEBS Letters, 2020, 594(20): 3243-3261. doi: 10.1002/1873-3468.13948
    [17] DE WINTER D A M, HSIEH C, MARKO M, et al. Cryo‐FIB preparation of whole cells and tissue for cryo‐TEM: use of high‐pressure frozen specimens in tubes and planchets[J]. Journal of Microscopy, 2021, 281(2): 125-137. doi: 10.1111/jmi.12943
    [18] CHANG I Y, RAHMAN M, HARNED A, et al. Cryo-fluorescence microscopy of high-pressure frozen C. elegans enables correlative FIB-SEM imaging of targeted embryonic stages in the intact worm[J]. Methods in Cell Biology, 2021, 162: 223-252.
    [19] Sartori A, Gatz R, Beck F, et al. Correlative microscopy: Bridging the gap between fluorescence light microscopy and cryo-electron tomography[J]. Journal of Structural Biology, 2007, 160(2): 135-145.
    [20] Schwartz CL, Sarbash VI, Ataullakhanov FI, et al. Cryo‐fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching[J]. Journal of microscopy, 2007, 227(2): 98-109.
    [21] Schorb M, Briggs JAG. Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity[J]. Ultramicroscopy, 2014, 143(Supplement C): 24-32.
    [22] LI W X, STEIN S C, GREGOR I, et al. Ultra-stable and versatile widefield cryo-fluorescence microscope for single-molecule localization with sub-nanometer accuracy[J]. Optics Express, 2015, 23(3): 3770-3783. doi: 10.1364/OE.23.003770
    [23] XU X J, XUE Y H, TIAN B Y, et al. Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy[J]. Science China Life Sciences, 2018, 61(11): 1312-1319. doi: 10.1007/s11427-018-9380-3
    [24] HUSSELS M, KONRAD A, BRECHT M. Confocal sample-scanning microscope for single-molecule spectroscopy and microscopy with fast sample exchange at cryogenic temperatures[J]. Review of Scientific Instruments, 2012, 83(12): 123706. doi: 10.1063/1.4769996
    [25] KUBA J, MITCHELS J, HOVORKA M, et al. Advanced cryo-tomography workflow developments–correlative microscopy, milling automation and cryo-lift-out[J]. Journal of Microscopy, 2021, 281(2): 112-124. doi: 10.1111/jmi.12939
    [26] JEONG D, KIM D. Recent developments in correlative super-resolution fluorescence microscopy and electron microscopy[J]. Molecules and Cells, 2022, 45(1): 41-50. doi: 10.14348/molcells.2021.5011
    [27] LE GROS M A, MCDERMOTT G, UCHIDA M, et al. High-aperture cryogenic light microscopy[J]. Journal of Microscopy, 2009, 235(1): 1-8. doi: 10.1111/j.1365-2818.2009.03184.x
    [28] GISKE A. CryoSTED microscopy: a new spectroscopic approach for improving the resolution of STED microscopy using low temperature[D]. Heidelberg: Universität Heidelberg, 2007.
    [29] WURM C A, SCHWARZ H, JANS D C, et al. Correlative STED super-resolution light and electron microscopy on resin sections[J]. Journal of Physics D:Applied Physics, 2019, 52(37): 374003. doi: 10.1088/1361-6463/ab2b31
    [30] PRABHAKAR N, PEURLA M, KOHO S, et al. STED‐TEM correlative microscopy leveraging nanodiamonds as intracellular dual‐contrast markers[J]. Small, 2018, 14(5): 1701807. doi: 10.1002/smll.201701807
    [31] ANDRIAN T, DELCANALE P, PUJALS S, et al. Correlating super-resolution microscopy and transmission electron microscopy reveals multiparametric heterogeneity in nanoparticles[J]. Nano Letters, 2021, 21(12): 5360-5368. doi: 10.1021/acs.nanolett.1c01666
    [32] GU L SH, LI Y Y, ZHANG SH W, et al. Molecular resolution imaging by repetitive optical selective exposure[J]. Nature Methods, 2019, 16(11): 1114-1118. doi: 10.1038/s41592-019-0544-2
    [33] GU L SH, LI Y Y, ZHANG SH W, et al. Molecular-scale axial localization by repetitive optical selective exposure[J]. Nature Methods, 2021, 18(4): 369-373. doi: 10.1038/s41592-021-01099-2
    [34] 周文超, 李政昊, 武杰. 单分子生物检测方法及应用研究进展[J]. 中国光学(中英文),2022,15(5):878-894.

    ZHOU W CH, LI ZH H, WU J. Research progress of single molecule biological detection methods and applications[J]. Chinese Optics, 2022, 15(5): 878-894. (in Chinese)
    [35] WOLFF G, HAGEN C, GRÜNEWALD K, et al. Towards correlative super-resolution fluorescence and electron cryo-microscopy[J]. Biology of the Cell, 2016, 108(9): 245-258. doi: 10.1111/boc.201600008
    [36] ROBICHAUX M A, POTTER V L, ZHANG ZH X, et al. Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy[J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(47): 23562-23572. doi: 10.1073/pnas.1902003116
    [37] MOSER F, PRAŽÁK V, MORDHORST V, et al. Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy[J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(11): 4804-4809. doi: 10.1073/pnas.1810690116
    [38] HOFFMAN D P, SHTENGEL G, XU C S, et al. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells[J]. Science, 2020, 367(6475): eaaz5357. doi: 10.1126/science.aaz5357
    [39] LIU B, XUE Y H, ZHAO W, et al. Three-dimensional super-resolution protein localization correlated with vitrified cellular context[J]. Scientific Reports, 2015, 5: 13017. doi: 10.1038/srep13017
    [40] PHILLIPS M A, HARKIOLAKI M, PINTO D M S, et al. CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging[J]. Optica, 2020, 7(7): 802-812. doi: 10.1364/OPTICA.393203
    [41] SCHERTEL A, KIRMSE R, HUMMEL E, et al.. Imaging of vitrified biological specimens by confocal cryo fluorescence microscopy and cryo FIB/SEM tomography[C]. European Microscopy Congress 2016. 2016.
    [42] ZACHS T, SCHERTEL A, MEDEIROS J, et al. Fully automated, sequential focused ion beam milling for cryo-electron tomography[J]. eLife, 2020, 9: e52286. doi: 10.7554/eLife.52286
    [43] WU G H, MITCHELL P G, GALAZ-MONTOYA J G, et al. Multi-scale 3D cryo-correlative microscopy for vitrified cells[J]. Structure, 2020, 28(11): 1231-1237.e3. doi: 10.1016/j.str.2020.07.017
    [44] LI SH G, JI G, SHI Y, et al. High-vacuum optical platform for cryo-CLEM (HOPE): a new solution for non-integrated multiscale correlative light and electron microscopy[J]. Journal of Structural Biology, 2018, 201(1): 63-75. doi: 10.1016/j.jsb.2017.11.002
    [45] FAAS F G A, BÁRCENA M, AGRONSKAIA A V, et al. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy[J]. Journal of Structural Biology, 2013, 181(3): 283-290. doi: 10.1016/j.jsb.2012.12.004
    [46] Optical path of the METEOR system. 2021. [EP/OL]. http://www.delmic.com/en/products/cryo-solutions/meteor
    [47] SMEETS M, BIEBER A, CAPITANIO C, et al. Integrated cryo-correlative microscopy for targeted structural investigation in situ[J]. Microscopy Today, 2021, 29(6): 20-25. doi: 10.1017/S1551929521001280
    [48] BIEBER A, CAPITANIO C, SCHIØTZ O, et al. Precise 3D-correlative FIB-milling of biological samples using METEOR, an integrated cryo-CLEM imaging system[J]. Microscopy and Microanalysis, 2021, 27(S1): 3230-3232. doi: 10.1017/S1431927621011132
    [49] SCHWARTZ C L, SARBASH V I, ATAULLAKHANOV F I, et al. Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching[J]. Journal of Microscopy, 2007, 227(2): 98-109. doi: 10.1111/j.1365-2818.2007.01794.x
    [50] DAHLBERG P D, MOERNER W E. Cryogenic super-resolution fluorescence and electron microscopy correlated at the nanoscale[J]. Annual Review of Physical Chemistry, 2021, 72: 253-278. doi: 10.1146/annurev-physchem-090319-051546
  • 加载中
图(6)
计量
  • 文章访问数:  658
  • HTML全文浏览量:  279
  • PDF下载量:  349
  • 被引次数: 0
出版历程
  • 收稿日期:  2022-05-10
  • 修回日期:  2022-06-14
  • 网络出版日期:  2022-10-11

目录

    /

    返回文章
    返回

    重要通知

    2024年2月16日科睿唯安通过Blog宣布,2024年将要发布的JCR2023中,229个自然科学和社会科学学科将SCI/SSCI和ESCI期刊一起进行排名!《中国光学(中英文)》作为ESCI期刊将与全球SCI期刊共同排名!