Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100–250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2–3 d, allowing for several pause points during the procedure.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Communications Physics Open Access 04 November 2023
Nature Methods Open Access 18 September 2023
TomoTwin: generalized 3D localization of macromolecules in cryo-electron tomograms with structural data mining
Nature Methods Open Access 15 May 2023
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Cryo-ET representative tomograms have been deposited in the Electron Microscopy Data Bank under the accession code EMD-21039.
Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State (Oxford Scholarship, 2010).
Nogales, E. & Scheres, S. H. W. H. W. Cryo-EM: a unique tool for the visualization of macromolecular complexity. Mol. Cell 58, 677–689 (2015).
Frank, J., ed. Electron Tomography: Methods for Three-Dimensional Visualization of Structures in the Cell (Springer, 2006).
Grimm, R., Typke, D., Bärmann, M. & Baumeister, W. Determination of the inelastic mean free path in ice by examination of tilted vesicles and automated most probable loss imaging. Ultramicroscopy 63, 169–179 (1996).
Pilhofer, M., Ladinsky, M. S., McDowall, A. W., Petroni, G. & Jensen, G. J. Microtubules in bacteria: ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton. PLoS Biol. 9, e1001213 (2011).
Beeby, M., Cho, M., Stubbe, J. & Jensen, G. J. Growth and localization of polyhydroxybutyrate granules in Ralstonia eutropha. J. Bacteriol. 194, 1092–1099 (2011).
Amat, F. et al. Analysis of the intact surface layer of Caulobacter crescentus by cryo-electron tomography. J. Bacteriol. 192, 5855–5865 (2010).
Szwedziak, P., Wang, Q., Bharat, T. A. M., Tsim, M. & Löwe, J. Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division. Elife 3, e04601 (2014).
Szwedziak, P., Wang, Q., Freund, S. M. V. & Löwe, J. FtsA forms actin-like protofilaments. EMBO J. 31, 2249–2260 (2012).
Hu, B., Lara-Tejero, M., Kong, Q., Galán, J. E. & Liu, J. In situ molecular architecture of the Salmonella type III secretion machine. Cell 168, 1065–1074.e10 (2017).
Jasnin, M. et al. Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails. Proc. Natl Acad. Sci. USA 110, 20521–20526 (2013).
Brandt, F., Carlson, L.-A., Hartl, F. U., Baumeister, W. & Grünewald, K. The three-dimensional organization of polyribosomes in intact human cells. Mol. Cell 39, 560–569 (2010).
Hanein, D. & Horwitz, A. R. The structure of cell–matrix adhesions: the new frontier. Curr. Opin. Cell Biol. 24, 134–140 (2012).
Asano, S. et al. Proteasomes. A molecular census of 26S proteasomes in intact neurons. Science 347, 439–442 (2015).
Strauss, M., Hofhaus, G., Schröder, R. R. & Kühlbrandt, W. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J. 27, 1154–1160 (2008).
Davies, K. M. et al. Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc. Natl Acad. Sci. USA 108, 14121–14126 (2011).
Davies, K. M., Blum, T. B. & Kühlbrandt, W. Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants. Proc. Natl Acad. Sci. USA 115, 3024–3029 (2018).
Kim, S. J. et al. Integrative structure and functional anatomy of a nuclear pore complex. Nature 555, 475–482 (2018).
Li, S., Fernandez, J.-J., Marshall, W. F. & Agard, D. A. Three-dimensional structure of basal body triplet revealed by electron cryo-tomography. EMBO J. 31, 552–562 (2011).
Bharat, T. A. M. et al. Structure of the immature retroviral capsid at 8A resolution by cryo- electron microscopy. Nature 487, 385–389 (2012).
Bui, K. H. H. et al. Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155, 1233–1243 (2013).
Pfeffer, S. et al. Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon. Nat. Commun. 5, 3072 (2014).
Mattei, S., Glass, B., Hagen, W. J. H., Kräusslich, H. G. & Briggs, J. A. G. The structure and flexibility of conical HIV-1 capsids determined within intact virions. Science 354, 1434–1437 (2016).
Schur, F. K. M. et al. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation. Science 353, 506–508 (2016).
Dodonova, S. O. et al. 9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments. Elife 6, e26691 (2017).
Beck, M. & Hurt, E. The nuclear pore complex: understanding its function through structural insight. Nat. Rev. Mol. Cell Biol. 18, 73–89 (2017).
Zuber, B. et al. Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state. J. Bacteriol. 190, 5672–5680 (2008).
Salje, J., Zuber, B. & Lowe, J. Electron cryomicroscopy of E. coli reveals filament bundles involved in plasmid DNA segregation. Science 323, 509–512 (2009).
Studer, D., Klein, A., Iacovache, I., Gnaegi, H. & Zuber, B. A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons. J. Struct. Biol. 185, 125–128 (2014).
Schur, F. K. M. et al. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution. Nature 517, 505–508 (2014).
Briggs, J. A. G. Structural biology in situ—the potential of subtomogram averaging. Curr. Opin. Struct. Biol. 23, 261–267 (2013).
Delarue, M. et al. mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell 174, 338–349.e20 (2018).
Turoňová, B., Schur, F. K. M., Wan, W. & Briggs, J. A. G. Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4Å. J. Struct. Biol. 199, 187–195 (2017).
Mosalaganti, S. et al. In situ architecture of the algal nuclear pore complex. Nat. Commun. 9, 2361 (2018).
Marko, M., Hsieh, C., Schalek, R., Frank, J. & Mannella, C. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat. Methods 4, 215–217 (2007).
Rigort, A. et al. Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc. Natl Acad. Sci. USA 109, 4449–4454 (2012).
Villa, E., Schaffer, M., Plitzko, J. M. & Baumeister, W. Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography. Curr. Opin. Struct. Biol. 23, 771–777 (2013).
Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J. & Gonen, T. Collection of continuous rotation microED data from ion beam-milled crystals of any size. Structure 27, 545–548.e2 (2019).
Medeiros, J. M. et al. Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography. Ultramicroscopy 190, 1–11 (2018).
de Winter, D. A. M. et al. In-situ integrity control of frozen-hydrated, vitreous lamellas prepared by the cryo-focused ion beam-scanning electron microscope. J. Struct. Biol. 183, 11–18 (2013).
Rigort, A. et al. Micromachining tools and correlative approaches for cellular cryo-electron tomography. J. Struct. Biol. 172, 169–179 (2010).
Hayles, M. F. et al. The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy. J. Struct. Biol. 172, 180–190 (2010).
Wang, K., Strunk, K., Zhao, G., Gray, J. L. & Zhang, P. 3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography. J. Struct. Biol. 180, 318–326 (2012).
Hsieh, C., Schmelzer, T., Kishchenko, G., Wagenknecht, T. & Marko, M. Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography. J. Struct. Biol. 185, 32–41 (2014).
Harapin, J. et al. Structural analysis of multicellular organisms with cryo-electron tomography. Nat. Methods 12, 634–636 (2015).
Zhang, J., Ji, G., Huang, X., Xu, W. & Sun, F. An improved cryo-FIB method for fabrication of frozen hydrated lamella. J. Struct. Biol. 194, 218–223 (2016).
Schaffer, M. et al. A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue. Nat. Methods 16, 757–762 (2019).
Rubino, S. et al. A site-specific focused-ion-beam lift-out method for cryo Transmission Electron Microscopy. J. Struct. Biol. 180, 572–576 (2012).
Wagenknecht, T., Hsieh, C. & Marko, M. Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography. Eur. J. Transl. Myol. 25, 49–56 (2015).
Mahamid, J. et al. A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms. J. Struct. Biol. 192, 262–269 (2015).
Khanna, K. et al. The molecular architecture of engulfment during Bacillus subtilis sporulation. Elife 8, e45257 (2019).
Chaikeeratisak, V. et al. Viral capsid trafficking along treadmilling tubulin filaments in bacteria. Cell 177, 1771–1780.e12 (2019).
Engel, B. D. et al. Correction: native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. Elife 4, e11383 (2015).
Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969–972 (2016).
Chaikeeratisak, V. et al. Assembly of a nucleus-like structure during viral replication in bacteria. Science 355, 194–197 (2017).
Lopez-Garrido, J. et al. Chromosome translocation inflates Bacillus forespores and impacts cellular morphology. Cell 172, 758–770.e14 (2018).
Hampton, C. M. et al. Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat. Protoc. 12, 150–167 (2017).
Arnold, J. et al. Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy. Biophys. J. 110, 860–869 (2016).
Watanabe, R. et al. The in situ structure of Parkinson’s disease-linked LRRK2 Preprint at https://www.biorxiv.org/content/10.1101/837203v1 (2019).
Guo, Q. et al. In situ structure of neuronal C9orf72 poly-GA aggregates reveals proteasome recruitment. Cell 172, 696–705.e12 (2018).
Bäuerlein, F. J. B. et al. In situ architecture and cellular interactions of polyQ inclusions. Cell 171, 179–187.e10 (2017).
Ader, N. R. et al. Molecular and topological reorganizations in mitochondrial architecture interplay during bax-mediated steps of apoptosis. Elife 8, e40712 (2019).
Weiss, G. L., Kieninger, A. K., Maldener, I., Forchhammer, K. & Pilhofer, M. Structure and function of a bacterial gap junction analog. Cell 178, 374–384.e15 (2019).
Rast, A. et al. Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane. Nat. Plants 5, 436–446 (2019).
Buckley, G. et al. Automated cryo-lamella preparation for high-throughput in-situ structural biology. J. Struct. Biol. 210, 107488 (2020).
Zachs, T. et al. Fully automated, sequential focused ion beam milling for cryo-electron tomography. Elife 9, e52286 (2020).
Nannenga, B. L. & Gonen, T. MicroED: a versatile cryoEM method for structure determination. Emerg. Top. Life Sci. 2, 1–8 (2018).
Lee, J. Z. et al. Cryogenic focused ion beam characterization of lithium metal anodes. ACS Energy Lett. 4, 489–493 (2019).
Zachman, M. J., Tu, Z., Choudhury, S., Archer, L. A. & Kourkoutis, L. F. Cryo-STEM mapping of solid-liquid interfaces and dendrites in lithium-metal batteries. Nature 560, 345–349 (2018).
Ladinsky, M. S. Micromanipulator-assisted vitreous cryosectioning and sample preparation by high-pressure freezing. Methods Enzymol. 481, 165–194 (2010).
Ladinsky, M. S., Pierson, J. M. & McIntosh, J. R. Vitreous cryo-sectioning of cells facilitated by a micromanipulator. J. Microsc. 224, 129–134 (2006).
Al-Amoudi, A., Norlen, L. P. O. & Dubochet, J. Cryo-electron microscopy of vitreous sections of native biological cells and tissues. J. Struct. Biol. 148, 131–135 (2004).
Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections. EMBO J. 23, 3583–3588 (2004).
Hsieh, C. E., Leith, A. D., Mannella, C. A., Frank, J. & Marko, M. Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections. J. Struct. Biol. 153, 1–13 (2006).
Bouchet-Marquis, C., Dubochet, J. & Fakan, S. Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture. Histochem. Cell Biol. 125, 43–51 (2006).
Matias, V. R. F., Al-Amoudi, A., Dubochet, J. & Beveridge, T. J. Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa. J. Bacteriol. 185, 6112–6118 (2003).
McEwen, B. F., Marko, M., Hsieh, C.-E. & Mannella, C. Use of frozen-hydrated axonemes to assess imaging parameters and resolution limits in cryoelectron tomography. J. Struct. Biol. 138, 47–57 (2002).
Pierson, J. et al. Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: electrostatic charging for section attachment and implementation of an anti-contamination glove box. J. Struct. Biol. 169, 219–225 (2010).
Larabell, C. A. & Le Gros, M. A. X-ray tomography generates 3-D reconstructions of the yeast, Saccharomyces cerevisiae, at 60-nm resolution. Mol. Biol. Cell 15, 957–962 (2004).
Schneider, G. et al. Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat. Methods 7, 985–987 (2010).
Hagen, C. et al. Structural basis of vesicle formation at the inner nuclear membrane. Cell 163, 1692–1701 (2015).
Hagen, C. et al. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J. Struct. Biol. 177, 193–201 (2012).
Hagen, C. et al. Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells. Ultramicroscopy 146, 46–54 (2014).
Wolf, S. G., Houben, L. & Elbaum, M. Cryo-scanning transmission electron tomography of vitrified cells. Nat. Methods 11, 423–428 (2014).
Wolf, S. G. et al. 3D visualization of mitochondrial solid-phase calcium stores in whole cells. Elife 6, e29929 (2017).
Schertel, A. et al. Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens. J. Struct. Biol. 184, 355–360 (2013).
Soto, G. E. et al. Serial section electron tomography: a method for three-dimensional reconstruction of large structures. Neuroimage 1, 230–243 (1994).
Heymann, J. A. W. et al. Site-specific 3D imaging of cells and tissues with a dual beam microscope. J. Struct. Biol. 155, 63–73 (2006).
Narayan, K. & Subramaniam, S. Focused ion beams in biology. Nat. Methods 12, 1021–1031 (2015).
Xu, C. S. et al. Enhanced FIB-SEM systems for large-volume 3D imaging. Elife 6, e25916 (2017).
Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004).
Helmstaedter, M. et al. Connectomic reconstruction of the inner plexiform layer in the mouse retina. Nature 500, 168–174 (2013).
Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).
Shu, X. et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).
Martell, J. D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).
Ou, H. D. et al. ChromEMT: visualizing 3D chromatin structure and compaction in interphase and mitotic cells. Science 357, eaag0025 (2017).
Sochacki, K. A., Shtengel, G., van Engelenburg, S. B., Hess, H. F. & Taraska, J. W. Correlative super-resolution fluorescence and metal-replica transmission electron microscopy. Nat. Methods 11, 305–308 (2014).
Han, Y. et al. Directed evolution of split APEX2 peroxidase. ACS Chem. Biol. 14, 619–635 (2019).
Ngo, J. T. et al. Click-EM for imaging metabolically tagged nonprotein biomolecules. Nat. Chem. Biol. 12, 459–465 (2016).
Ariotti, N. et al. Ultrastructural localisation of protein interactions using conditionally stable nanobodies. PLoS Biol. 16, e2005473 (2018).
Ariotti, N., Hall, T. E. & Parton, R. G. Correlative light and electron microscopic detection of GFP-labeled proteins using modular APEX. Methods Cell Biol. 140, 105–121 (2017).
Parton, R. G. Twenty years of traffic: a 2020 vision of cellular electron microscopy. Traffic 21, 4–5 (2019).
Toro-Nahuelpan, M. et al. Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies. Nat. Methods 17, 50–54 (2019).
Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228 (1988).
Taylor, K. A. & Glaeser, R. M. Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future. J. Struct. Biol. 163, 214–223 (2008).
Schaffer, M. et al. Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. J. Struct. Biol. 197, 73–82 (2017).
Wolff, G. et al. Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae. J. Struct. Biol. 208, 107389 (2019).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Mastronarde, D. N. SerialEM: a program for automated tilt series acquisition on Tecnai microscopes using prediction of specimen position. Microsc. Microanal. 9, 1182–1183 (2003).
Hagen, W. J. H., Wan, W. & Briggs, J. A. G. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–198 (2017).
Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).
We thank Julia Mahamid, Bernd Fruhberger and Villa lab members for insightful discussions and technical support. The subtomogram average of ribosome shown in Fig. 8 was performed by Robert Buschauer. This work was supported by an NIH Director’s New Innovator Award 1DP2GM123494-01 and the National Science Foundation MRI grant NSF DBI 1920374. We acknowledge the use of the UC San Diego cryo-Electron Microscopy Facility (partially supported by a gift from the Agouron Institute to UC San Diego) and the San Diego Nanotechnology Infrastructure of UC San Diego, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (ECCS-1542148). D.S. is supported by the Damon Runyon Cancer Research Foundation (DRG-#2364-19).
F.R.W., R.W., D.S., M.S., J.P. and E.V. have no competing interests. R.S. and H.P. are employees of TFS, and P.F. was an employee.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Chaikeeratisak, V. et al. Science 355, 194–197 (2017): https://science.sciencemag.org/content/355/6321/194.long
Chaikeeratisak, V. et al. Cell 177, 1771–1780.e12 (2019): https://www.sciencedirect.com/science/article/pii/S0092867419305604
Khanna, K. et al. eLife 8, e45257 (2019): https://elifesciences.org/articles/45257
Watanabe, R. et al. Preprint at https://www.biorxiv.org/content/10.1101/837203v1 (2019)
About this article
Cite this article
Wagner, F.R., Watanabe, R., Schampers, R. et al. Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography. Nat Protoc 15, 2041–2070 (2020). https://doi.org/10.1038/s41596-020-0320-x
This article is cited by
Communications Physics (2023)
Nature Methods (2023)
Nature Communications (2023)