DOI: 10.1073/pnas.1201333109. Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

10.1073/pnas.1201333109

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

Bibliography

Rigort, A., BÃ¤uerlein, F. J. B., Villa, E., Eibauer, M., Laugks, T., Baumeister, W., & Plitzko, J. M. (2012). Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proceedings of the National Academy of Sciences, 109(12), 4449â4454.

Authors 7

Alexander Rigort (first)
Felix J. B. Bäuerlein (additional)
Elizabeth Villa (additional)
Matthias Eibauer (additional)
Tim Laugks (additional)
Wolfgang Baumeister (additional)
Jürgen M. Plitzko (additional)

References 33 Referenced 408

Dates

Type	When
Created	13 years, 5 months ago (March 5, 2012, 11:33 p.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 9:16 p.m.)
Indexed	1 day, 14 hours ago (Aug. 19, 2025, 6:59 a.m.)
Issued	13 years, 5 months ago (March 5, 2012)
Published	13 years, 5 months ago (March 5, 2012)
Published Online	13 years, 5 months ago (March 5, 2012)
Published Print	13 years, 5 months ago (March 20, 2012)

Funders 0

None

BibTeX

@article{Rigort_2012, title={Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography}, volume={109}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.1201333109}, DOI={10.1073/pnas.1201333109}, number={12}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Rigort, Alexander and Bäuerlein, Felix J. B. and Villa, Elizabeth and Eibauer, Matthias and Laugks, Tim and Baumeister, Wolfgang and Plitzko, Jürgen M.}, year={2012}, month=mar, pages={4449–4454} }

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