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          Institute: MPI für molekulare Zellbiologie und Genetik     Collection: MPI-CBG Publications 2015 (arch)     Display Documents



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ID: 718027.0, MPI für molekulare Zellbiologie und Genetik / MPI-CBG Publications 2015 (arch)
A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms.
Authors:Mahamid, J.; Schampers, Ruud; Persoon, Hans; Hyman, Anthony; Baumeister, W.; Plitzko, Jürgen M
Date of Publication (YYYY-MM-DD):2015
Title of Journal:Journal of Structural Biology
Volume:192
Issue / Number:2
Start Page:262
End Page:269
Copyright:not available
Audience:Experts Only
Intended Educational Use:No
Abstract / Description: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.
External Publication Status:published
Document Type:Article
Version Comment:Automatic journal name synchronization
Communicated by:Thüm
Affiliations:MPI für molekulare Zellbiologie und Genetik
Identifiers:LOCALID:6296
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