Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.

Department of Molecular Structural Biology Max Planck Institute of Biochemistry Martinsried Germany

Leica Microsystems CMS GmbH Mannheim Germany

Leica Microsystems GmbH Vienna Austria

Thermo Fisher Scientific Brno s r o Brno Czech Republic

Thermo Fisher Scientific FEI Deutschland GmbH Planegg Germany

Zobrazit více v PubMed

Ader, N.R. & Kukulski, W. (2017) triCLEM: combining high-precision, room temperature CLEM with cryo-fluorescence microscopy to identify very rare events. Methods Cell Biol. 140, 303-320.

Albert, S., Wietrzynski, W., Lee, C.W. et al. (2020) Direct visualization of degradation microcompartments at the ER membrane. Proc. Natl. Acad. Sci. U.S.A. 117(2), 1069-1080.

Arnold, J., Mahamid, J., Lucic, V. et al. (2016) Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy. Biophys. J. 110(4), 860-9.

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

Brüggeller, P. & Mayer, E. (1980) Complete vitrification in pure liquid water and dilute aqueous solutions. Nature 288(5791), 569-571.

Buckley, G., Gervinskas, G., Taveneau, C., Venugopal, H., Whisstock, J.C. & de Marco, A. (2020) Automated cryo-lamella preparation for high-throughput in-situ structural biology. J. Struct. Biol. 210, 107488.

Chang, Y.W., Chen, S., Tocheva, E.I. et al. (2014) Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat. Methods 11(7), 737-739.

Dubochet, J., Adrian, M., Chang, J.J. et al. (1988) Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21(2), 129-228.

Dubochet, J. & McDowall, A.W. (1981) Vitrification of pure water for electron microscopy. J. Microsc. 124(3), 3-4.

Faas, F.G., Barcena, M., Agronskaia, A.V. et al. (2013) Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy. J. Struct. Biol. 181(3), 283-290.

Faoro, R., Bassu, M., Mejia, Y.X. et al. (2018) Aberration-corrected cryoimmersion light microscopy. Proc. Natl. Acad. Sci. U.S.A. 115(6), 1204-1209.

Fokkema, J., Fermie, J., Liv, N. et al. (2018) Fluorescently labelled silica coated gold nanoparticles as fiducial markers for correlative light and electron microscopy. Sci. Rep. 8(1), 13625.

Gorelick, S., Buckley, G., Gervinskas, G. et al. (2019) PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. Elife 8, e45919. https://doi.org/10.7554/eLife.45919.

Guo, Q., Lehmer, C., Martinez-Sanchez, A. et al. (2018) In Situ structure of neuronal C9orf72 Poly-GA aggregates reveals proteasome recruitment. Cell 172(4), 696-705 e12.

Hampton, C.M., Strauss, J.D., Ke, Z. et al. (2017) Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat. Protoc. 12(1), 150-167.

Hayles, M.F., Stokes, D.J., Phifer, D. & Findlay, K.C. (2007) A technique for improved focused ion beam milling of cryo-prepared life science specimens. J. Microsc. 226(Pt 3), 263-269.

Heymann, J.A., Hayles, M., Gestmann, I., Giannuzzi, L.A., Lich, B. & Subramaniam, S. (2006) Site-specific 3D imaging of cells and tissues with a dual beam microscope. J. Struct Biol. 155(1), 63-73.

Kaufmann, R., Hagen, C. & Grunewald, K.. (2014) Fluorescence cryo-microscopy: current challenges and prospects. Curr. Opin. Chem. Biol. 20, 86-91.

Kaufmann, R., Schellenberger, P., Seiradake, E. et al. (2014) Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions. Nano Lett. 14(7), 4171-4175.

Koning, R.I., Celler, K., Willemse, J., Bos, E., van Wezel, G.P. & Koster, A.J. (2014) Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces. Methods Cell Biol. 124, 217-239.

Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. & Briggs, J.A. (2011) Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J. Cell. Biol. 192(1), 111-119.

Langford, R.M. & Clinton, C. (2004) In situ lift-out using a FIB-SEM system. Micron 35(7), 607-611.

Langford, R.M., Huang, Y.Z., Lozano-Perez, S., Titchmarsh, J.M. & Petford-Long, A.K. (2001) Preparation of site specific transmission electron microscopy plan-view specimens using a focused ion beam system. J. Vacuum Sci. Technol. B: Microelectr. Nanometer Struct. Process., Measure., Phenom. 19(3), 755-758.

Le Gros, M.A., McDermott, G., Uchida, M., Knoechel, C.G. & Larabell, C.A. (2009) High-aperture cryogenic light microscopy. J. Microsc. 235(1), 1-8.

Liu, B., Xue, Y., Zhao, W. et al. (2015) Three-dimensional super-resolution protein localization correlated with vitrified cellular context. Sci. Rep. 5, 13017.

Mahamid, J., Pfeffer, S., Schaffer, M. et al. (2016) Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351(6276), 969-672.

Mahamid, J., Schampers, R., Persoon, H., Hyman, A.A., Baumeister, W. & Plitzko, J.M. (2015) A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms. J. Struct. Biol. 192(2), 262-269.

Marko, M., Hsieh, C., Moberlychan, W., Mannella, C.A. & Frank, J. (2006) Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples. J. Microsc. 222(Pt 1), 42-47.

Marko, M., Hsieh, C., Schalek, R., Frank, J. & Mannella, C. (2007) Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat. Methods 4(3), 215-217.

Mosalaganti, S., Kosinski, J., Albert, S. et al. (2018) In situ architecture of the algal nuclear pore complex. Nat. Commun. 9(1), 2361.

Moser, F., Prazak, V., Mordhorst, V. et al. (2019) Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy. Proc. Natl. Acad. Sci. U.S.A. 116(11), 4804-4809.

Nahmani, M., Lanahan, C., DeRosier, D. & Turrigiano, G.G. (2017) High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions. Proc. Natl. Acad. Sci. U.S.A. 114(15), 3832-3836.

Nievergelt, A.P., Viar, G.A. & Pigino, G. (2019) Towards a mechanistic understanding of cellular processes by cryoEM. Curr. Opin. Struct. Biol. 58, 149-158.

Parmenter, C.D., Fay, M.W., Hartfield, C. & Eltaher, H.M. (2016) Making the practically impossible “Merely difficult” - Cryogenic FIB lift-out for “Damage free” soft matter imaging. Microsc. Res. Tech. 79(4), 298-303.

Plitzko, J. & Baumeister, W.P. (2019) Cryo-electron tomography. Springer Handbook of Microscopy (ed. by P.W. Hawkes & J.C.H. Spence), p. 2. Springer International Publishing, Cham.

Reymann, J. (2018) Lightning - Image Information Extraction by Adaptive Deconvolution. Leica Microsystems White Paper. 1-13. https://downloads.leica-microsystems.com/LIGHTNING/Publications/LIGHTNING_Whitepaper_v_Mar2020.pdf.

Rigort, A., Bauerlein, F.J., Leis, A. et al. (2010) Micromachining tools and correlative approaches for cellular cryo-electron tomography. J. Struct. Biol. 172(2), 169-179.

Rigort, A., Bauerlein, F.J., Villa, E. et al. (2012) Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc. Natl. Acad. Sci. U.S.A. 109(12), 4449-4454.

Rigort, A. & Plitzko, JM. (2015) Cryo-focused-ion-beam applications in structural biology. Arch. Biochem. Biophys. 581, 122-130.

Rubino, S., Akhtar, S., Melin, P., Searle, A., Spellward, P. & Leifer, K. (2012) A site-specific focused-ion-beam lift-out method for cryo Transmission Electron Microscopy. J. Struct. Biol. 180(3), 572-576.

Sartori, A., Gatz, R., Beck, F., Rigort, A., Baumeister, W. & Plitzko, J.M. (2007) Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J. Struct. Biol. 160(2), 135-145.

Schaffer, M., Engel, B.D., Laugks, T., Mahamid, J., Plitzko, J.M. & Baumeister, W. (2015) Cryo-focused ion beam sample preparation for imaging vitreous cells by cryo-electron tomography. Bio. Protoc. 5(17), e1575.

Schaffer, M., Mahamid, J., Engel, B.D., Laugks, T., Baumeister, W. & Plitzko, J.M. (2017) Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. J. Struct. Biol. 197(2), 73-82.

Schaffer, M., Pfeffer, S., Mahamid, J. et al. (2019) A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue. Nat. Methods 16(8), 757-762.

Schellenberger, P., Kaufmann, R., Siebert, C.A., Hagen, C., Wodrich, H. & Grunewald, K.. (2014) High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers. Ultramicroscopy 143, 41-51.

Schertel, A., Snaidero, N., Han, H.M. et al. (2013) Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens. J. Struct. Biol. 184(2), 355-360.

Schorb, M. & Briggs, JA. (2014) Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity. Ultramicroscopy 143, 24-32.

Schorb, M., Gaechter, L., Avinoam, O. et al. (2017) New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography. J. Struct. Biol. 197(2), 83-93.

Schorb, M. & Sieckmann, F. (2017) Matrix MAPS - an intuitive software to acquire, analyze, and annotate light microscopy data for CLEM. Methods Cell Biol. 140, 321-333.

Schumacher, J. & Bertrand, L. (2019) THUNDER Imagers: How Do They Really Work? Leica Microsystems Technology Note. 1-10. www.leica-microsystems.com/science-lab/thunder-technology-note/.

Schwartz, C.L., Sarbash, V.I., Ataullakhanov, F.I., McIntosh, J.R. & Nicastro, D. (2007) Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J. Microsc. 227(Pt 2), 98-109.

Tacke, S., Erdmann, P., Wang, Z. et al. (2020) A streamlined workflow for automated cryo focused ion beam milling. bioRxiv. https://doi.org/10.1101/2020.02.24.96303.

Tuijtel, M.W., Koster, A.J., Jakobs, S., Faas, F.G.A. & Sharp, T.H. (2019) Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins. Sci. Rep. 9(1), 1369.

van Driel, L.F., Valentijn, J.A., Valentijn, K.M., Koning, R.I. & Koster, A.J. (2009) Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells. Eur. J. Cell Biol. 88(11), 669-684.

van Hest, J.J.H.A., Agronskaia, A.V., Fokkema, J. et al. (2019) Towards robust and versatile single nanoparticle fiducial markers for correlative light and electron microscopy. J. Microsc. 274(1), 13-22.

Wagner, J., Schaffer, M. & Fernandez-Busnadiego, R. (2017) Cryo-electron tomography - the cell biology that came in from the cold. FEBS Lett. 591(17), 2520-2533.

Wan, W. & Briggs, J.A. (2016) Cryo-electron tomography and subtomogram averaging. Methods Enzymol. 579, 329-367.

Wang, L., Bateman, B., Zanetti-Domingues, L.C. et al. (2019) Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution. Commun. Biol. 2, 74.

Wolff, G., Hagen, C., Grunewald, K. & Kaufmann, R. (2016) Towards correlative super-resolution fluorescence and electron cryo-microscopy. Biol. Cell 108(9), 245-258.

Wolff, G., Limpens, R., Zheng, S. et al. (2019) Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae. J. Struct. Biol. 208(3), 107389.

Zachman, M.J., Asenath-Smith, E., Estroff, L.A. & Kourkoutis, L.F. (2016) Site-specific preparation of intact solid-liquid interfaces by label-free in situ localization and cryo-focused ion beam lift-Out. Microsc. Microanal. 22(6), 1338-1349.

Zachs, T., Schertel, A., Medeiros, J. et al. (2020) Fully automated, sequential focused ion beam milling for cryo-electron tomography. Elife 9, e52286. https://doi.org/10.7554/eLife.52286.

Zhang, P. (2019) Advances in cryo-electron tomography and subtomogram averaging and classification. Curr. Opin. Struct. Biol. 58, 249-258.

Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ Cryo-ET