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Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography

Nature Protocols volume 15, pages 2041–2070 (2020)Cite this article

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Abstract

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.

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Fig. 1: Key steps of the procedure described in the protocol.
Fig. 2: Blotting and clipping grids into FIB-AutoGrids.
Fig. 3: SEM-FIB geometry in dual-beam microscopy.
Fig. 4: Cryo-stage setup.
Fig. 5: The sample preparation station.
Fig. 6: The transfer rod is used for sample transfer under vacuum.
Fig. 7: Various procedures/considerations for milling.
Fig. 8: Representative data of the cellular cryo-ET using the protocol described here.
Fig. 9: Issues occurring during cryo-FIB milling that require optimization of the workflow.

Data availability

Cryo-ET representative tomograms have been deposited in the Electron Microscopy Data Bank under the accession code EMD-21039.

References

  1. Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State (Oxford Scholarship, 2010).

  2. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Frank, J., ed. Electron Tomography: Methods for Three-Dimensional Visualization of Structures in the Cell (Springer, 2006).

  4. 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).

    Article  CAS  PubMed  Google Scholar 

  5. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Beeby, M., Cho, M., Stubbe, J. & Jensen, G. J. Growth and localization of polyhydroxybutyrate granules in Ralstonia eutropha. J. Bacteriol. 194, 1092–1099 (2011).

    Article  PubMed  CAS  Google Scholar 

  7. Amat, F. et al. Analysis of the intact surface layer of Caulobacter crescentus by cryo-electron tomography. J. Bacteriol. 192, 5855–5865 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Szwedziak, P., Wang, Q., Freund, S. M. V. & Löwe, J. FtsA forms actin-like protofilaments. EMBO J. 31, 2249–2260 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jasnin, M. et al. Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails. Proc. Natl Acad. Sci. USA 110, 20521–20526 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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).

    Article  CAS  PubMed  Google Scholar 

  13. Hanein, D. & Horwitz, A. R. The structure of cell–matrix adhesions: the new frontier. Curr. Opin. Cell Biol. 24, 134–140 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Asano, S. et al. Proteasomes. A molecular census of 26S proteasomes in intact neurons. Science 347, 439–442 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kim, S. J. et al. Integrative structure and functional anatomy of a nuclear pore complex. Nature 555, 475–482 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Bharat, T. A. M. et al. Structure of the immature retroviral capsid at 8A resolution by cryo- electron microscopy. Nature 487, 385–389 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Bui, K. H. H. et al. Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155, 1233–1243 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Pfeffer, S. et al. Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon. Nat. Commun. 5, 3072 (2014).

    Article  PubMed  CAS  Google Scholar 

  23. 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).

    Article  CAS  PubMed  Google Scholar 

  24. 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).

    Article  CAS  PubMed  Google Scholar 

  25. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Beck, M. & Hurt, E. The nuclear pore complex: understanding its function through structural insight. Nat. Rev. Mol. Cell Biol. 18, 73–89 (2017).

    Article  CAS  PubMed  Google Scholar 

  27. Zuber, B. et al. Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state. J. Bacteriol. 190, 5672–5680 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Salje, J., Zuber, B. & Lowe, J. Electron cryomicroscopy of E. coli reveals filament bundles involved in plasmid DNA segregation. Science 323, 509–512 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. 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).

    Article  PubMed  Google Scholar 

  30. 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).

    Article  PubMed  CAS  Google Scholar 

  31. Briggs, J. A. G. Structural biology in situ—the potential of subtomogram averaging. Curr. Opin. Struct. Biol. 23, 261–267 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Delarue, M. et al. mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell 174, 338–349.e20 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. 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).

    Article  CAS  PubMed  Google Scholar 

  36. Rigort, A. et al. Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc. Natl Acad. Sci. USA 109, 4449–4454 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 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).

    Article  CAS  PubMed  Google Scholar 

  38. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 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).

    Article  CAS  PubMed  Google Scholar 

  40. 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).

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  42. 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).

    Article  PubMed  Google Scholar 

  43. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 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).

    Article  CAS  PubMed  Google Scholar 

  45. Harapin, J. et al. Structural analysis of multicellular organisms with cryo-electron tomography. Nat. Methods 12, 634–636 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. 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).

    Article  PubMed  Google Scholar 

  47. 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).

    Article  CAS  PubMed  Google Scholar 

  48. 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).

    Article  PubMed  Google Scholar 

  49. 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).

    Article  PubMed  Google Scholar 

  50. 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).

    Article  CAS  PubMed  Google Scholar 

  51. Khanna, K. et al. The molecular architecture of engulfment during Bacillus subtilis sporulation. Elife 8, e45257 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Chaikeeratisak, V. et al. Viral capsid trafficking along treadmilling tubulin filaments in bacteria. Cell 177, 1771–1780.e12 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Engel, B. D. et al. Correction: native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. Elife 4, e11383 (2015).

    Article  PubMed Central  Google Scholar 

  54. Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969–972 (2016).

    Article  CAS  PubMed  Google Scholar 

  55. Chaikeeratisak, V. et al. Assembly of a nucleus-like structure during viral replication in bacteria. Science 355, 194–197 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Lopez-Garrido, J. et al. Chromosome translocation inflates Bacillus forespores and impacts cellular morphology. Cell 172, 758–770.e14 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 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).

    Article  CAS  PubMed  Google Scholar 

  58. Arnold, J. et al. Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy. Biophys. J. 110, 860–869 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 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).

  60. Guo, Q. et al. In situ structure of neuronal C9orf72 poly-GA aggregates reveals proteasome recruitment. Cell 172, 696–705.e12 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Bäuerlein, F. J. B. et al. In situ architecture and cellular interactions of polyQ inclusions. Cell 171, 179–187.e10 (2017).

    Article  PubMed  CAS  Google Scholar 

  62. Ader, N. R. et al. Molecular and topological reorganizations in mitochondrial architecture interplay during bax-mediated steps of apoptosis. Elife 8, e40712 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  63. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Rast, A. et al. Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane. Nat. Plants 5, 436–446 (2019).

    Article  CAS  PubMed  Google Scholar 

  65. Buckley, G. et al. Automated cryo-lamella preparation for high-throughput in-situ structural biology. J. Struct. Biol. 210, 107488 (2020).

    Article  CAS  PubMed  Google Scholar 

  66. Zachs, T. et al. Fully automated, sequential focused ion beam milling for cryo-electron tomography. Elife 9, e52286 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Nannenga, B. L. & Gonen, T. MicroED: a versatile cryoEM method for structure determination. Emerg. Top. Life Sci. 2, 1–8 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Lee, J. Z. et al. Cryogenic focused ion beam characterization of lithium metal anodes. ACS Energy Lett. 4, 489–493 (2019).

    Article  CAS  Google Scholar 

  69. 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).

    Article  CAS  PubMed  Google Scholar 

  70. Ladinsky, M. S. Micromanipulator-assisted vitreous cryosectioning and sample preparation by high-pressure freezing. Methods Enzymol. 481, 165–194 (2010).

    Article  CAS  PubMed  Google Scholar 

  71. Ladinsky, M. S., Pierson, J. M. & McIntosh, J. R. Vitreous cryo-sectioning of cells facilitated by a micromanipulator. J. Microsc. 224, 129–134 (2006).

    Article  PubMed  Google Scholar 

  72. 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).

    Article  CAS  PubMed  Google Scholar 

  73. Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections. EMBO J. 23, 3583–3588 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. 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).

    Article  CAS  PubMed  Google Scholar 

  75. 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).

    Article  CAS  PubMed  Google Scholar 

  76. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. 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).

    Article  PubMed  Google Scholar 

  78. 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).

    Article  PubMed  Google Scholar 

  79. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Schneider, G. et al. Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat. Methods 7, 985–987 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Hagen, C. et al. Structural basis of vesicle formation at the inner nuclear membrane. Cell 163, 1692–1701 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Hagen, C. et al. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J. Struct. Biol. 177, 193–201 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  83. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Wolf, S. G., Houben, L. & Elbaum, M. Cryo-scanning transmission electron tomography of vitrified cells. Nat. Methods 11, 423–428 (2014).

    Article  CAS  PubMed  Google Scholar 

  85. Wolf, S. G. et al. 3D visualization of mitochondrial solid-phase calcium stores in whole cells. Elife 6, e29929 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  86. Schertel, A. et al. Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens. J. Struct. Biol. 184, 355–360 (2013).

    Article  CAS  PubMed  Google Scholar 

  87. Soto, G. E. et al. Serial section electron tomography: a method for three-dimensional reconstruction of large structures. Neuroimage 1, 230–243 (1994).

    Article  CAS  PubMed  Google Scholar 

  88. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  89. Narayan, K. & Subramaniam, S. Focused ion beams in biology. Nat. Methods 12, 1021–1031 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Xu, C. S. et al. Enhanced FIB-SEM systems for large-volume 3D imaging. Elife 6, e25916 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  91. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  92. Helmstaedter, M. et al. Connectomic reconstruction of the inner plexiform layer in the mouse retina. Nature 500, 168–174 (2013).

    Article  CAS  PubMed  Google Scholar 

  93. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    Article  CAS  PubMed  Google Scholar 

  94. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Martell, J. D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Ou, H. D. et al. ChromEMT: visualizing 3D chromatin structure and compaction in interphase and mitotic cells. Science 357, eaag0025 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  97. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Han, Y. et al. Directed evolution of split APEX2 peroxidase. ACS Chem. Biol. 14, 619–635 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Ngo, J. T. et al. Click-EM for imaging metabolically tagged nonprotein biomolecules. Nat. Chem. Biol. 12, 459–465 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Ariotti, N. et al. Ultrastructural localisation of protein interactions using conditionally stable nanobodies. PLoS Biol. 16, e2005473 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  101. 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).

    Article  PubMed  Google Scholar 

  102. Parton, R. G. Twenty years of traffic: a 2020 vision of cellular electron microscopy. Traffic 21, 4–5 (2019).

    Google Scholar 

  103. Toro-Nahuelpan, M. et al. Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies. Nat. Methods 17, 50–54 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  104. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228 (1988).

    Article  CAS  PubMed  Google Scholar 

  105. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. 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).

    Article  CAS  PubMed  Google Scholar 

  107. 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).

    Article  PubMed  Google Scholar 

  108. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  109. 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).

    Article  Google Scholar 

  110. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  111. 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).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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).

Author information

Author notes

  1. Felix R. Wagner

    Present address: Department of Molecular Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany

  2. Peter Fruhstorfer

    Present address: Eppendorf AG, Hamburg, Germany

  3. These authors contributed equally: Felix R. Wagner, Reika Watanabe.

Authors and Affiliations

  1. Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA

    Felix R. Wagner, Reika Watanabe, Digvijay Singh & Elizabeth Villa

  2. Thermo Fisher Scientific, Eindhoven, the Netherlands

    Ruud Schampers, Hans Persoon & Peter Fruhstorfer

  3. Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany

    Miroslava Schaffer & Jürgen Plitzko

Authors
  1. Felix R. Wagner
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  2. Reika Watanabe
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  3. Ruud Schampers
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  4. Digvijay Singh
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  5. Hans Persoon
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  6. Miroslava Schaffer
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  7. Peter Fruhstorfer
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  8. Jürgen Plitzko
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  9. Elizabeth Villa
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Contributions

F.R.W., R.W., D.S. and E.V. designed the project. F.R.W., R.W., R.S., D.S. and E.V. performed the experiments. R.S., H.P. and E.V. designed and built components of the system. F.R.W., R.W., D.S. and E.V. wrote the manuscript with input from the other authors.

Corresponding author

Correspondence to Elizabeth Villa.

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Competing interests

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.

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Related links

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)

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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

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