Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Integrated fluorescence light microscopy-guided cryo-focused ion beam-milling for in situ montage cryo-ET

Nature Protocols (2026)Cite this article

Subjects

Abstract

Cryogenic-electron tomography (cryo-ET) permits the in situ visualization of biological macromolecules at the molecular level. Owing to the variable thickness of cells, tissues and organisms, frozen specimens may need to be thinned by cryo-focused ion beam (FIB) milling to produce thin (<500 nm) cryo-lamellae suitable for cryo-ET. Locating regions of interest remains a challenge because untargeted milling can lead to inadvertent ablation and removal of regions of interest. Correlative light and electron microscopy, combined with cryo-FIB milling, can guide the identification of labeled targets in the cellular milieu. Multiple transfers between cryo-imaging instruments, cumbersome correlation algorithms, limited accuracy and low throughput have hindered the routine adoption of cryo-FIB milling within a multimodal correlative workflow for in situ structural biology. Here we present a workflow for 3D correlative cryo-fluorescence light microscopy-FIB-ET that streamlines fluorescence light microscopy-guided FIB milling, improving throughput while preserving both structural and contextual information. The complete integration of hardware and software described here minimizes sample contamination from cross-platform exchanges and greatly enhances the efficiency of 3D targeting in cryo-milling. We then describe procedures for implementing montage parallel array cryo-ET (MPACT), which can be easily adapted to any modern life-science transmission electron microscope. MPACT supports high-throughput cryo-ET acquisitions (10 tilt series in 1.5 h) for structure determination and comprehensive contextual understanding of macromolecules within their native surroundings. A complete session from sample preparation to MPACT data processing takes 5−7 d for an individual experienced in both cryo-EM and cryo-FIB milling.

Key points

  • Coupling 3D correlative focused ion beam milling with montage cryo-electron tomography enables the precise localization of regions of interest deep within cells and the acquisition of larger fields of view that preserve high-resolution structural details.

  • Single-pair region of interest-marker registration, combined with integrated software and hardware, enhances correlative milling workflows. Montage parallel array cryo-electron tomography produces montages and individual tile tomograms, thereby eliminating possible mechanical adjustments to the transmission electron microscope.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview for iFLM-guided cryo-FIB-milling for in situ montage cryo-ET.
Fig. 2: Flowchart of the steps for 3D-cryo-iFLM-FIB-MPACT protocol.
Fig. 3: Configuration of iFLM in a cryo-FIB-SEM Aquilos 2 system.
Fig. 4: Transmittance performance and improved resolution of FLM imaging under cryogenic conditions.
Fig. 5: Coordinate-based single ROI–marker pair 3D correlation in an iFLM-FIB-SEM system.
Fig. 6: Grid optimization and vitrification.
Fig. 7: Movement of fiducial markers during cryo-FIB milling.
Fig. 8: Representative results of iFLM-guided FIB-milling and MPACT of HeLa cells to study LD–mitochondrial contact sites.
Fig. 9: Adaptability of the iFLM-guided FIB milling-cryo-ET workflow.
Fig. 10: TomoGrapher GUI to enable optimizations of MPACT acquisition schemes via 3D simulation.
Fig. 11: MPACT acquisition in SerialEM 4.1.
Fig. 12: MAPS software for tile set collection and FLM image alignment.
Fig. 13: Main software packages (iFLM, MAPS and AutoTEM) for the streamlined single-pair ROI–marker FLM-guided automated FIB milling.
Fig. 14: Representative segmentation analysis of distinct morphologies of actin-like filaments near the LD–mitochondrial site captured by MPACT.

Data availability

All relevant data are available from the corresponding author upon request. The iFLM image stacks and corresponding SEM and FIB images are associated with the on-the-fly 3D correlation procedure. While it is challenging to share, a demonstration could be performed to help the user upon reasonable request. A set of raw frames for a representative 3 × 3 MPACT montage tilt series is provided as a tutorial dataset and available to download at https://github.com/wright-cemrc-projects/cryoet-montage/tree/main/Tutorial/ for demonstration of MPACT preprocessing, stitching and individual tile tilt series generation. Subtomogram averages of respiratory syncytial virus fusion pair particles using MPACT individual tile tilt series have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-40308 and EMD-40307.

Code availability

CorRelator and TomoGrapher are open-source software, and the source code can be downloaded via https://github.com/wright-cemrc-projects. The MPACT processing scripts can be downloaded via https://github.com/wright-cemrc-projects/cryoet-montage/tree/main/Bashscripts.

References

  1. Hong, Y., Song, Y., Zhang, Z. & Li, S. Cryo-electron tomography: the resolution revolution and a surge of in situ virological discoveries. Annu. Rev. Biophys. 52, 339–360 (2023).

    Article  CAS  PubMed  Google Scholar 

  2. Turk, M. & Baumeister, W. The promise and the challenges of cryo-electron tomography. FEBS Lett. 594, 3243–3261 (2020).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Berger, C. et al. Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology. Nat. Methods 20, 499–511 (2023).

    Article  CAS  PubMed  Google Scholar 

  5. 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 

  6. 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 

  7. Yang, J. E., Larson, M. R., Sibert, B. S., Shrum, S. & Wright, E. R. CorRelator: interactive software for real-time high precision cryo-correlative light and electron microscopy. J. Struct. Biol. 213, 107709 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Smeets, M. et al. Integrated cryo-correlative microscopy for targeted structural investigation in situ. Microsc. Today 29, 20–25 (2021).

    Article  Google Scholar 

  9. 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 

  10. Hall, A. S., Lavery, L. L. & Doux, P. Effective multimodal multiscale analytical and imaging correlation. IEEE Sens. Lett. 3, 1–4 (2019).

    Article  Google Scholar 

  11. Moser, F. et al. Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy. Proc. Natl Acad. Sci. USA 116, 4804–4809 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Chang, Y.-W. et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat. Methods 11, 737–739 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fukuda, Y. et al. Coordinate transformation based cryo-correlative methods for electron tomography and focused ion beam milling. Ultramicroscopy 143, 15–23 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Kelley, K. et al. Waffle Method: a general and flexible approach for improving throughput in FIB-milling. Nat. Commun. 13, 1857 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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 

  17. Booy, F. P. & Pawley, J. B. Cryo-crinkling: what happens to carbon films on copper grids at low temperature. Ultramicroscopy 48, 273–280 (1993).

    Article  CAS  PubMed  Google Scholar 

  18. Li, W. et al. Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation. Nat. Methods 20, 268–275 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li, S. et al. ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study. Nat. Methods 20, 276–283 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gorelick, S. et al. PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. eLife 8, e45919 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Tacke, S. et al. A streamlined workflow for automated cryo focused ion beam milling. J. Struct. Biol. 213, 107743 (2021).

    Article  CAS  PubMed  Google Scholar 

  22. Klumpe, S. et al. A modular platform for automated cryo-FIB workflows. eLife 10 (2021).

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

  24. Hylton, R. K. & Swulius, M. T. Challenges and triumphs in cryo-electron tomography. iScience 24, 102959 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Schur, F. K. M. Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging. Curr. Opin. Struct. Biol. 58, 1–9 (2019).

    Article  CAS  PubMed  Google Scholar 

  26. Yang, J. E. et al. Correlative montage parallel array cryo-tomography for in situ structural cell biology. Nat. Methods 20, 1537–1543 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huang, Y.-W., Cambre, M. & Lee, H.-J. The toxicity of nanoparticles depends on multiple molecular and physicochemical mechanisms. Int. J. Mol. Sci. 18, 2702 (2017).

  28. Scher, N., Rechav, K., Paul-Gilloteaux, P. & Avinoam, O. In situ fiducial markers for 3D correlative cryo-fluorescence and FIB-SEM imaging. iScience 24, 102714 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yang, J. E., Mitchell, J. M., Bingman, C. A., Mosher, D. F. & Wright, E. R. In situ crystalline structure of the human eosinophil major basic protein-1. Preprint at bioRxiv https://doi.org/10.1101/2024.10.09.617336 (2024).

  30. Kim, J. Y. et al. Handling difficult cryo-ET samples: a study with primary neurons from Drosophila melanogaster. Microsc. Microanal. 29, 2127–2148 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Sibert, B. S., Kim, J. Y., Yang, J. E. & Wright, E. R. Micropatterning transmission electron microscopy grids to direct cell positioning within whole-cell cryo-electron tomography workflows. J. Vis. Exp. 175, e62992 (2021).

  32. Sibert, B. S. et al. Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers. Nat. Commun. 15, 5923 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  34. Mastronarde, D. Tomographic reconstruction with the IMOD software package. Microsc. Microanal. 12, 178–179 (2006).

    Article  Google Scholar 

  35. Garza-Lopez, E. et al. Protocols for generating surfaces and measuring 3D organelle morphology using Amira. Cells 11, 65 (2022).

  36. Stalling, D., Westerhoff, M. & Hege, H.-C. in The Visualization Handbook (Academic Press, 2005).

  37. Chen, M. et al. Convolutional neural networks for automated annotation of cellular cryo-electron tomograms. Nat. Methods 14, 983–985 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Heebner, J. E. et al. Deep learning-based segmentation of cryo-electron tomograms. J. Vis. Exp. 189, e64435 (2022).

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

    Article  CAS  PubMed  Google Scholar 

  40. Castaño-Díez, D., Kudryashev, M., Arheit, M. & Stahlberg, H. Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J. Struct. Biol. 178, 139–151 (2012).

    Article  PubMed  Google Scholar 

  41. Nicastro, D. et al. The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313, 944–948 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Bharat, T. A. M. & Scheres, S. H. W. Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION. Nat. Protoc. 11, 2054–2065 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wagner, F. R. et al. Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography. Nat. Protoc. 15, 2041–2070 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Boltje, D. B. et al. A cryogenic, coincident fluorescence, electron, and ion beam microscope. eLife 11, e82891 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Schiøtz, O. H. et al. Serial Lift-Out: sampling the molecular anatomy of whole organisms. Nat. Methods 21, 1684–1692 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Paul-Gilloteaux, P. et al. eC-CLEM: flexible multidimensional registration software for correlative microscopies. Nat. Methods 14, 102–103 (2017).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  48. Kukulski, W. et al. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J. Cell Biol. 192, 111–119 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Peck, A. et al. Montage electron tomography of vitrified specimens. J. Struct. Biol. 214, 107860 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Elferich, J., Schiroli, G., Scadden, D. T. & Grigorieff, N. Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections. eLife 11, e80980 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Chua, E. Y. D. et al. Square beams for optimal tiling in transmission electron microscopy. Nat. Methods 21, 562–565 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Brown, H. G., Smith, D., Wardle, B. C. & Hanssen, E. Square condenser apertures for square cameras in low-dose transmission electron microscopy. Nat. Methods 21, 566–568 (2024).

    Article  CAS  PubMed  Google Scholar 

  53. Eisenstein, F. et al. Parallel cryo electron tomography on in situ lamellae. Nat. Methods 20, 131–138 (2023).

    Article  CAS  PubMed  Google Scholar 

  54. Bouvette, J. et al. Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography. Nat. Commun. 12, 1957 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Eisenstein, F., Danev, R. & Pilhofer, M. Improved applicability and robustness of fast cryo-electron tomography data acquisition. J. Struct. Biol. 208, 107–114 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Chreifi, G., Chen, S., Metskas, L. A., Kaplan, M. & Jensen, G. J. Rapid tilt-series acquisition for electron cryotomography. J. Struct. Biol. 205, 163–169 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Sexton, D. L., Burgold, S., Schertel, A. & Tocheva, E. I. Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans. Curr. Res. Struct. Biol. 4, 1–9 (2022).

    Article  CAS  PubMed  Google Scholar 

  58. Weber, N., Hinks, B., Jensen, J., Lidahl, T. & Mendonça, L. Sample preparation for in situ cryotomography of mammalian cells. J. Vis. Exp. 202, e65697 (2023).

  59. Thompson, R. F., Walker, M., Siebert, C. A., Muench, S. P. & Ranson, N. A. An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology. Methods 100, 3–15 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. de Beer, M. et al. Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP. Commun. Biol. 6, 510 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Toro-Nahuelpan, M. et al. Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies. Nat. Methods 17, 50–54 (2020).

    Article  CAS  PubMed  Google Scholar 

  62. Bäuerlein, F. J. B. et al. Cryo-electron tomography of large biological specimens vitrified by plunge freezing. Preprint at bioRxiv https://doi.org/10.1101/2021.04.14.437159 (2023).

  63. Hands-Portman, I. & Bakker, S. E. Customising the plunge-freezing workflow for challenging conditions. Faraday Discuss. 240, 44–54 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  65. Metskas, L. A. & Briggs, J. A. G. Fluorescence-based detection of membrane fusion state on a cryo-EM grid using correlated cryo-fluorescence and cryo-electron microscopy. Microsc. Microanal. 25, 942–949 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Karuppasamy, M., Karimi Nejadasl, F., Vulovic, M., Koster, A. J. & Ravelli, R. B. Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate. J. Synchrotron Radiat. 18, 398–412 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Basanta, B., Hirschi, M. M., Grotjahn, D. A. & Lander, G. C. A case for glycerol as an acceptable additive for single-particle cryoEM samples. Acta Crystallogr. D. 78, 124–135 (2022).

    Article  CAS  Google Scholar 

  68. Melo, R. C. N. & Weller, P. F. Contemporary understanding of the secretory granules in human eosinophils. J. Leukoc. Biol. 104, 85–93 (2018).

    Article  CAS  PubMed  Google Scholar 

  69. Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  70. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Chen, M. et al. A complete data processing workflow for cryo-ET and subtomogram averaging. Nat. Methods 16, 1161–1168 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Tegunov, D., Xue, L., Dienemann, C., Cramer, P. & Mahamid, J. Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells. Nat. Methods 18, 186–193 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Dimchev, G., Amiri, B., Fäßler, F., Falcke, M. & Schur, F. K. Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data. J. Struct. Biol. 213, 107808 (2021).

    Article  PubMed  Google Scholar 

  74. Lam, V. & Villa, E. Practical approaches for cryo-FIB milling and applications for cellular cryo-electron tomography. Methods Mol. Biol. 2215, 49–82 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Rigort, A. et al. Automated segmentation of electron tomograms for a quantitative description of actin filament networks. J. Struct. Biol. 177, 135–144 (2012).

    Article  CAS  PubMed  Google Scholar 

  76. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  77. 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 

  78. Langford, R. M. Focused ion beam nanofabrication: a comparison with conventional processing techniques. J. Nanosci. Nanotechnol. 6, 661–668 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful for the TEM and SEM instrumentation support of M. Woods and T. Coomes from Thermo Fisher Scientific. We are thankful for the computational resources supplied through the SBGrid76. This work was supported in part by the University of Wisconsin, Madison, the Department of Biochemistry at the University of Wisconsin, Madison, the Morgridge Institute for Research, and public health service grant nos. R01 GM114561, R01 GM132068, RF1 NS110436 and U24 GM139168 to E.R.W., P01 HL088594 to N. Jarjour, and R01 AI125390 to D.F.M. and J. Coon from the National Institutes of Health. J.M.M. was supported by the grant T32 HL07899 from the National Heart, Lung, and Blood Institute, National Institutes of Health. This work was supported in part by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under award number DE-SC0018409. We are grateful for the use of facilities and instrumentation at the Cryo-EM Research Center and the Midwest Center for Cryo-ET in the Department of Biochemistry at the University of Wisconsin, Madison.

Author information

Author notes

  1. Joshua M. Mitchell

    Present address: Investigative Medicine, Homer Stryker M.D. School of Medicine, Western Michigan University, Kalamazoo, MI, USA

Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin, Madison, WI, USA

    Jie E. Yang, Bryan S. Sibert, Matt R. Larson & Elizabeth R. Wright

  2. Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, USA

    Jie E. Yang, Bryan S. Sibert, Matt R. Larson & Elizabeth R. Wright

  3. Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA

    Jie E. Yang, Bryan S. Sibert, Matt R. Larson & Elizabeth R. Wright

  4. Thermo Fisher Scientific Brno, Brno, Czech Republic

    Veronika Vrbovská & John Mitchels

  5. Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA

    Joshua M. Mitchell & Deane F. Mosher

  6. Department of Medicine, University of Wisconsin, Madison, WI, USA

    Joshua M. Mitchell

  7. Thermo Fisher Scientific Planegg, Bavaria, Germany

    Tilman Franke & Alex Rigort

  8. Thermo Fisher Scientific, Houston, TX, USA

    Alexander S. Hall & Deane F. Mosher

  9. Morgridge Institute for Research, Madison, WI, USA

    Elizabeth R. Wright

  10. DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI, USA

    Elizabeth R. Wright

  11. Investigative Medicine, Homer Stryker M.D. School of Medicine, Western Michigan University, Kalamazoo, MI, USA

    Deane F. Mosher

Authors
  1. Jie E. Yang
    View author publications

    Search author on:PubMed Google Scholar

  2. Veronika Vrbovská
    View author publications

    Search author on:PubMed Google Scholar

  3. Joshua M. Mitchell
    View author publications

    Search author on:PubMed Google Scholar

  4. Tilman Franke
    View author publications

    Search author on:PubMed Google Scholar

  5. Bryan S. Sibert
    View author publications

    Search author on:PubMed Google Scholar

  6. Matt R. Larson
    View author publications

    Search author on:PubMed Google Scholar

  7. Alexander S. Hall
    View author publications

    Search author on:PubMed Google Scholar

  8. Alex Rigort
    View author publications

    Search author on:PubMed Google Scholar

  9. Deane F. Mosher
    View author publications

    Search author on:PubMed Google Scholar

  10. John Mitchels
    View author publications

    Search author on:PubMed Google Scholar

  11. Elizabeth R. Wright
    View author publications

    Search author on:PubMed Google Scholar

Contributions

E.R.W., J.M., J.E.Y., V.V., T.F. and A.R. conceived the study. J.E.Y. and E.R.W. designed the study. J.E.Y., V.V., B.S.S., J.M.M., T.F., A.S.H., A.R., M.R.L., D.F.M., J.M. and E.R.W. prepared the samples, performed the experiments and processed the data. J.E.Y. and E.R.W. wrote the manuscript with contributions from all authors. All authors read and approved the manuscript.

Corresponding author

Correspondence to Elizabeth R. Wright.

Ethics declarations

Competing interests

V.V., T.F., A.H., A.R. and J.M. are employees of Thermo Fisher Scientific. Other authors have no competing interests or other interests that might be perceived to influence the interpretation of the article.

Peer review

Peer review information

Nature Protocols thanks Johannes Groen, Michael Martynowycz and Anna Steyer for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Key references

Hampton, C. M. et al. Nat. Protoc. 12, 150–167 (2017): https://doi.org/10.1038/nprot.2016.168

Yang, J. E. et al. J. Struct. Biol. 213, 107709 (2021): https://doi.org/10.1016/j.jsb.2021.107709

Sibert, B. S. et al. J. Vis. Exp. 175, 10.3791/62992 (2021): https://doi.org/10.3791/62992

Yang, J. E. et al. Nat. Methods 20, 1537 (2023): https://doi.org/10.1038/s41592-023-01999-5

Kim, J. Y. et al. Microsc. Microanal. 29, 2127 (2023): https://doi.org/10.1093/micmic/ozad125

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, J.E., Vrbovská, V., Mitchell, J.M. et al. Integrated fluorescence light microscopy-guided cryo-focused ion beam-milling for in situ montage cryo-ET. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01284-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • DOI: https://doi.org/10.1038/s41596-025-01284-z

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing