title: Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography.
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.
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Rigort
authors.forename: Felix J B
authors.initials: FJ
authors.lastname: Bäuerlein
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Villa
authors.forename: Matthias
authors.initials: M
authors.lastname: Eibauer
authors.forename: Tim
authors.initials: T
authors.lastname: Laugks
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D016616:Cryoultramicrotomy; D004023:Dictyostelium; D004583:Electrons; D004867:Equipment Design; D005057:Eukaryotic Cells / Q000166:cytology*; D005583:Fourier Analysis; D005615:Freezing; D007477:Ions; D046911:Macromolecular Substances / Q000737:chemistry; D008855:Microscopy, Electron, Scanning / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D013696:Temperature; D014057:Tomography, X-Ray Computed / Q000379:methods*; D058989:Vitrification
chemical_list: D007477:Ions; D046911:Macromolecular Substances