*Result*: Integrated fluorescence light microscopy-guided cryo-focused ion beam-milling for in situ montage cryo-ET.
Title:
Integrated fluorescence light microscopy-guided cryo-focused ion beam-milling for in situ montage cryo-ET.
Authors:
Yang JE; Department of Biochemistry, University of Wisconsin, Madison, WI, USA.; Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, USA.; Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA., Vrbovská V; Thermo Fisher Scientific Brno, Brno, Czech Republic., Mitchell JM; Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA.; Department of Medicine, University of Wisconsin, Madison, WI, USA.; Investigative Medicine, Homer Stryker M.D. School of Medicine, Western Michigan University, Kalamazoo, MI, USA., Franke T; Thermo Fisher Scientific Planegg, Bavaria, Germany., Sibert BS; Department of Biochemistry, University of Wisconsin, Madison, WI, USA.; Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, USA.; Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA., Larson MR; Department of Biochemistry, University of Wisconsin, Madison, WI, USA.; Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, USA.; Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA., Hall AS; Thermo Fisher Scientific, Houston, TX, USA., Rigort A; Thermo Fisher Scientific Planegg, Bavaria, Germany., Mosher DF; Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA.; Thermo Fisher Scientific, Houston, TX, USA.; Investigative Medicine, Homer Stryker M.D. School of Medicine, Western Michigan University, Kalamazoo, MI, USA., Mitchels J; Thermo Fisher Scientific Brno, Brno, Czech Republic., Wright ER; Department of Biochemistry, University of Wisconsin, Madison, WI, USA. erwright2@wisc.edu.; Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, USA. erwright2@wisc.edu.; Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, USA. erwright2@wisc.edu.; Morgridge Institute for Research, Madison, WI, USA. erwright2@wisc.edu.; DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI, USA. erwright2@wisc.edu.
Source:
Nature protocols [Nat Protoc] 2026 Jan 30. Date of Electronic Publication: 2026 Jan 30.
Publication Model:
Ahead of Print
Publication Type:
Journal Article; Review
Language:
English
Journal Info:
Publisher: Nature Pub. Group Country of Publication: England NLM ID: 101284307 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1750-2799 (Electronic) Linking ISSN: 17502799 NLM ISO Abbreviation: Nat Protoc Subsets: MEDLINE
Imprint Name(s):
Original Publication: London, UK : Nature Pub. Group, 2006-
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Rigort, A. et al. Micromachining tools and correlative approaches for cellular cryo-electron tomography. J. Struct. Biol. 172, 169–179 (2010). (PMID: 2017884810.1016/j.jsb.2010.02.011)
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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). (PMID: 33610654860140510.1016/j.jsb.2021.107709)
Smeets, M. et al. Integrated cryo-correlative microscopy for targeted structural investigation in situ. Microsc. Today 29, 20–25 (2021). (PMID: 10.1017/S1551929521001280)
Arnold, J. et al. Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy. Biophys. J. 110, 860–869 (2016). (PMID: 26769364477585410.1016/j.bpj.2015.10.053)
Hall, A. S., Lavery, L. L. & Doux, P. Effective multimodal multiscale analytical and imaging correlation. IEEE Sens. Lett. 3, 1–4 (2019). (PMID: 10.1109/LSENS.2018.2878667)
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). (PMID: 30808803642140410.1073/pnas.1810690116)
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). (PMID: 27368127528735510.1016/j.jsb.2016.06.020)
Chang, Y.-W. et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat. Methods 11, 737–739 (2014). (PMID: 24813625408147310.1038/nmeth.2961)
Fukuda, Y. et al. Coordinate transformation based cryo-correlative methods for electron tomography and focused ion beam milling. Ultramicroscopy 143, 15–23 (2014). (PMID: 2433246210.1016/j.ultramic.2013.11.008)
Kelley, K. et al. Waffle Method: a general and flexible approach for improving throughput in FIB-milling. Nat. Commun. 13, 1857 (2022). (PMID: 35387991898709010.1038/s41467-022-29501-3)
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). (PMID: 3153677410.1016/j.jsb.2019.09.006)
Booy, F. P. & Pawley, J. B. Cryo-crinkling: what happens to carbon films on copper grids at low temperature. Ultramicroscopy 48, 273–280 (1993). (PMID: 847559710.1016/0304-3991(93)90101-3)
Li, W. et al. Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation. Nat. Methods 20, 268–275 (2023). (PMID: 36646896991135310.1038/s41592-022-01749-z)
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). (PMID: 36646897991135110.1038/s41592-022-01748-0)
Gorelick, S. et al. PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. eLife 8, e45919 (2019). (PMID: 31259689660933310.7554/eLife.45919)
Tacke, S. et al. A streamlined workflow for automated cryo focused ion beam milling. J. Struct. Biol. 213, 107743 (2021). (PMID: 3397128610.1016/j.jsb.2021.107743)
Klumpe, S. et al. A modular platform for automated cryo-FIB workflows. eLife 10 (2021).
Zachs, T. et al. Fully automated, sequential focused ion beam milling for cryo-electron tomography. eLife 9 (2020).
Hylton, R. K. & Swulius, M. T. Challenges and triumphs in cryo-electron tomography. iScience 24, 102959 (2021). (PMID: 34466785838300610.1016/j.isci.2021.102959)
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). (PMID: 3100575410.1016/j.sbi.2019.03.018)
Yang, J. E. et al. Correlative montage parallel array cryo-tomography for in situ structural cell biology. Nat. Methods 20, 1537–1543 (2023). (PMID: 377232451055582310.1038/s41592-023-01999-5)
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).
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). (PMID: 34258551825396710.1016/j.isci.2021.102714)
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).
Kim, J. Y. et al. Handling difficult cryo-ET samples: a study with primary neurons from Drosophila melanogaster. Microsc. Microanal. 29, 2127–2148 (2023). (PMID: 379669781116823610.1093/micmic/ozad125)
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Grant Information:
R01 GM132068 United States GM NIGMS NIH HHS; RF1 NS110436 United States NS NINDS NIH HHS; U24 GM139168 United States GM NIGMS NIH HHS; T32 HL07899 U.S. Department of Health & Human Services | National Institutes of Health (NIH); R01 AI125390 United States AI NIAID NIH HHS; DE-SC0018409 U.S. Department of Energy (DOE)
Entry Date(s):
Date Created: 20260130 Latest Revision: 20260130
Update Code:
20260131
DOI:
10.1038/s41596-025-01284-z
PMID:
41618010
Database:
MEDLINE
*Further Information*
*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.
(© 2026. Springer Nature Limited.)*
*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.*