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SCIENCE CHINA Life Sciences, Volume 61, Issue 11: 1312-1319(2018) https://doi.org/10.1007/s11427-018-9380-3

Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy

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  • ReceivedAug 14, 2018
  • AcceptedSep 17, 2018
  • PublishedNov 2, 2018

Abstract

Remarkable progress in correlative light and electron cryo-microscopy (cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope (cryo-FM). Here, we describe an ultra-stable super-resolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than 0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system (average single molecule localization accuracy of ~13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy (SMLM) and cryogenic super-resolution correlative light and electron microscopy (csCLEM).


Funded by

the National Key R&D Program of China(2016YFA05002032016YFA05024002017YFA05047002017YFA0505300)

the National Natural Science Foundation of China(3166114304131127901)

Joint Program between Chinese Academy of Sciences and Peking University.


Acknowledgment

This work was supported by the National Key R&D Program of China (2016YFA0500203, 2016YFA0502400, 2017YFA0504700, 2017YFA0505300), the National Natural Science Foundation of China (31661143041, 31127901) and Joint Program between Chinese Academy of Sciences and Peking University.


Interest statement

The author (s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Calibration curve used for obtaining the axial position.

Figure S2 Preparation of the cryo-section.

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


References

[1] Bellare J.R., Davis H.T., Scriven L.E., Talmon Y.. Controlled environment vitrification system: an improved sample preparation technique. J Elec Microsc Tech, 1988, 10: 87-111 CrossRef PubMed Google Scholar

[2] Betzig E., Patterson G.H., Sougrat R., Lindwasser O.W., Olenych S., Bonifacino J.S., Davidson M.W., Lippincott-Schwartz J., Hess H.F.. Imaging intracellular fluorescent proteins at nanometer resolution. Science, 2006, 313: 1642-1645 CrossRef PubMed ADS Google Scholar

[3] Bleck C.K.E., Merz A., Gutierrez M.G., Walther P., Dubochet J., Zuber B., Griffiths G.. Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis. J Microscopy, 2010, 237: 23-38 CrossRef PubMed Google Scholar

[4] Briegel, A., Chen, S., Koster, A.J., Plitzko, J.M., Schwartz, C.L., and Jensen, G.J. (2010). Correlated light and electron cryo-microscopy. Methods Enzymol 481, 317–341. Google Scholar

[5] Chang H., Zhang M., Ji W., Chen J., Zhang Y., Liu B., Lu J., Zhang J., Xu P., Xu T.. A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications. Proc Natl Acad Sci USA, 2012, 109: 4455-4460 CrossRef PubMed ADS Google Scholar

[6] Chang Y.W., Chen S., Tocheva E.I., Treuner-Lange A., Löbach S., Søgaard-Andersen L., Jensen G.J.. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat Methods, 2014, 11: 737-739 CrossRef PubMed Google Scholar

[7] de Boer, P., Hoogenboom, J.P., and Giepmans, B.N. (2015). Correlated light and electron microscopy: ultrastructure lights up! Nat Methods 12, 503–513. Google Scholar

[8] Dubochet J.. Cryo-EM-the first thirty years. J Microscopy, 2012, 245: 221-224 CrossRef Google Scholar

[9] Dubochet J., Adrian M., Chang J.J., Homo J.C., Lepault J., McDowall A.W., Schultz P.. Cryo-electron microscopy of vitrified specimens. Quart Rev Biophys, 1988, 21: 129-228 CrossRef Google Scholar

[10] Glaeser, R.M. (2016). How good can cryo-EM become? Nat Methods 13, 28–32. Google Scholar

[11] Hess S.T., Girirajan T.P.K., Mason M.D.. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J, 2006, 91: 4258-4272 CrossRef ADS Google Scholar

[12] Hirschfeld, V., and Hubner, C.G. (2010). A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K. Rev Sci Instrum 81, 113705. Google Scholar

[13] Huang B., Wang W., Bates M., Zhuang X.. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science, 2008, 319: 810-813 CrossRef PubMed ADS Google Scholar

[14] Hurbain I., Sachse M.. The future is cold: cryo-preparation methods for transmission electron microscopy of cells. Biol Cell, 2011, 103: 405-420 CrossRef PubMed Google Scholar

[15] Hussels, M., Konrad, A., and Brecht, M. (2012). Confocal sample-scanning microscope for single-molecule spectroscopy and microscopy with fast sample exchange at cryogenic temperatures. Rev Sci Instrum 83, 123706. Google Scholar

[16] Kaufmann R., Hagen C., Grünewald K.. Fluorescence cryo-microscopy: current challenges and prospects. Curr Opin Chem Biol, 2014, 20: 86-91 CrossRef PubMed Google Scholar

[17] Kaufmann R., Schellenberger P., Seiradake E., Dobbie I.M., Jones E.Y., Davis I., Hagen C., Grünewald K.. Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions. Nano Lett, 2014, 14: 4171-4175 CrossRef PubMed ADS Google Scholar

[18] Kozankiewicz B., Orrit M.. Single-molecule photophysics, from cryogenic to ambient conditions. Chem Soc Rev, 2014, 43: 1029-1043 CrossRef PubMed Google Scholar

[19] Le Gros M.A., McDermott G., Uchida M., Knoechel C.G., Larabell C.A.. High-aperture cryogenic light microscopy. J Microscopy, 2009, 235: 1-8 CrossRef PubMed Google Scholar

[20] Li W., Stein S.C., Gregor I., Enderlein J.. Ultra-stable and versatile widefield cryo-fluorescence microscope for single-molecule localization with sub-nanometer accuracy. Opt Express, 2015, 23: 3770-3783 CrossRef ADS Google Scholar

[21] Liu B., Xue Y., Zhao W., Chen Y., Fan C., Gu L., Zhang Y., Zhang X., Sun L., Huang X., et al. Three-dimensional super-resolution protein localization correlated with vitrified cellular context. Sci Rep, 2015, 5: 13017 CrossRef PubMed ADS Google Scholar

[22] McDonald K.L.. A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues. J Microscopy, 2009, 235: 273-281 CrossRef PubMed Google Scholar

[23] Müller-Reichert, T., and Verkade, P. (2014). Preface. Correlative light and electron microscopy II. Methods Cell Biol 124, xvii–xviii. Google Scholar

[24] Peddie C.J., Blight K., Wilson E., Melia C., Marrison J., Carzaniga R., Domart M.C., O'Toole P., Larijani B., Collinson L.M.. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells. Ultramicroscopy, 2014, 143: 3-14 CrossRef PubMed Google Scholar

[25] Perkovic M., Kunz M., Endesfelder U., Bunse S., Wigge C., Yu Z., Hodirnau V.V., Scheffer M.P., Seybert A., Malkusch S., et al. Correlative light- and electron microscopy with chemical tags. J Struct Biol, 2014, 186: 205-213 CrossRef PubMed Google Scholar

[26] Rodriguez J.A., Ivanova M.I., Sawaya M.R., Cascio D., Reyes F.E., Shi D., Sangwan S., Guenther E.L., Johnson L.M., Zhang M., et al. Structure of the toxic core of α-synuclein from invisible crystals. Nature, 2015, 525: 486-490 CrossRef PubMed ADS Google Scholar

[27] Rust M.J., Bates M., Zhuang X.. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Meth, 2006, 3: 793-796 CrossRef PubMed Google Scholar

[28] Sartori A., Gatz R., Beck F., Rigort A., Baumeister W., Plitzko J.M.. Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J Struct Biol, 2007, 160: 135-145 CrossRef PubMed Google Scholar

[29] Schwartz C.L., Sarbash V.I., Ataullakhanov F.I., McIntosh J.R., Nicastro D.. Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J Microsc, 2007, 227: 98-109 CrossRef PubMed Google Scholar

[30] Wang S., Li S., Ji G., Huang X., Sun F.. Using integrated correlative cryo-light and electron microscopy to directly observe syntaphilin-immobilized neuronal mitochondria in situ. Biophys Rep, 2017, 3: 8-16 CrossRef PubMed Google Scholar

[31] Weinhausen, B., Saldanha, O., Wilke, R.N., Dammann, C., Priebe, M., Burghammer, M., Sprung, M., and Koster, S. (2014). Scanning X-ray nanodiffraction on living eukaryotic cells in microfluidic environments. Phys Rev Lett 112, 202–209. Google Scholar

[32] Weisenburger, S., Jing, B., Renn, A., and Sandoghdar, V. (2013). Cryogenic localization of single molecules with angstrom precision. Nanoimag Nanospectrosc 8815, 27. Google Scholar

[33] Wolff G., Hagen C., Grünewald K., Kaufmann R.. Towards correlative super-resolution fluorescence and electron cryo-microscopy. Biol Cell, 2016, 108: 245-258 CrossRef PubMed Google Scholar

[34] Zhang, Y.D., Gu, L.S., Chang, H., Ji, W., Chen, Y., Zhang, M.S., Yang, L., Liu, B., Chen, L.Y., and Xu, T. (2013). Ultrafast, accurate, and robust localization of anisotropic dipoles. Protein Cell 4, 598–606. Google Scholar

[35] Zondervan R., Kulzer F., Kol'chenk M.A., Orrit M.. Photobleaching of rhodamine 6G in poly(vinyl alcohol) at the ensemble and single-molecule levels. J Phys Chem A, 2004, 108: 1657-1665 CrossRef ADS Google Scholar

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