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  A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms

Mahamid, J., Schampers, R., Persoon, H., Hyman, A. A., Baumeister, W., & Plitzko, J. M. (2015). A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms. JOURNAL OF STRUCTURAL BIOLOGY, 192(2), 262-269. doi:10.1016/j.jsb.2015.07.012.

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 Creators:
Mahamid, Julia1, Author              
Schampers, Ruud2, Author
Persoon, Hans2, Author
Hyman, Anthony A.2, Author
Baumeister, Wolfgang1, Author              
Plitzko, Jürgen M.1, Author              
Affiliations:
1Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565142              
2external, ou_persistent22              

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Free keywords: TRANSMISSION ELECTRON-MICROSCOPY; CRYOELECTRON TOMOGRAPHY; CAENORHABDITIS-ELEGANS; MOLECULAR LANDSCAPES; BIOLOGICAL SAMPLES; VITREOUS SECTIONS; EUKARYOTIC CELLS; MAMMALIAN-CELLS; PHASE PLATE; IN-SITUCryo-SEM; Cryo-FIB; Cryo-TEM; Cryo-planing; Cryo-FLM; Correlative microscopy; Sample preparation;
 Abstract: Cryo-electron tomography provides 3D views of cellular architecture with molecular resolution. A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. Recently it has been shown that cryo-focused ion beam milling of plunge-frozen eukaryotic cells can produce homogeneously thin, distortion free lamellas for cryo-electron tomography. Multicellular organisms and tissue cannot be properly vitrified and thinned using this technique because they are considerably thicker. High pressure freezing is therefore necessary to provide optimal preservation. Here, we describe a workflow for preparing lamellas from Caenorhabditis elegans worms using cryo-FIB applied to high pressure frozen samples. We employ cryo-planing followed by correlative cryo-fluorescence microscopy to navigate this large multicellular volume and to localize specific targets within. To produce vitreous lamellas amenable to cryo-TEM observations at these targeted locations, we have developed a dedicated lift-out procedure at cryogenic temperature. (C) 2015 Elsevier Inc. All rights reserved.

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Language(s): eng - English
 Dates: 2015
 Publication Status: Published in print
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000364607400017
DOI: 10.1016/j.jsb.2015.07.012
 Degree: -

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Title: JOURNAL OF STRUCTURAL BIOLOGY
  Other : Special Issue: Recent Advances in Detector Technologies and Applications for Molecular TEM
Source Genre: Journal
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Publ. Info: 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA : ACADEMIC PRESS INC ELSEVIER SCIENCE
Pages: - Volume / Issue: 192 (2) Sequence Number: - Start / End Page: 262 - 269 Identifier: ISSN: 1047-8477