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

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Mahamid,  Julia
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Baumeister,  Wolfgang
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Plitzko,  Jürgen M.
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0029-35F4-3
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.