title: A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms.
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.
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany. Electronic address: Mahamid@biochem.mpg.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Mahamid
authors.affiliation: FEI Company Eindhoven, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands.
authors.forename: Ruud
authors.initials: R
authors.lastname: Schampers
authors.affiliation: FEI Company Eindhoven, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Persoon
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Hyman
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany. Electronic address: Plitzko@biochem.mpg.de.
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D005629:Frozen Sections / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D058989:Vitrification
keywords: Correlative microscopy; Cryo-FIB; Cryo-FLM; Cryo-SEM; Cryo-TEM; Cryo-planing; Sample preparation