title: Micromachining tools and correlative approaches for cellular cryo-electron tomography.
abstract: A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography.
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, 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: Andrew
authors.initials: A
authors.lastname: Leis
authors.forename: Manuela
authors.initials: M
authors.lastname: Gruska
authors.forename: Christian
authors.initials: C
authors.lastname: Hoffmann
authors.forename: Tim
authors.initials: T
authors.lastname: Laugks
authors.forename: Ulrike
authors.initials: U
authors.lastname: Böhm
authors.forename: Matthias
authors.initials: M
authors.lastname: Eibauer
authors.forename: Helmut
authors.initials: H
authors.lastname: Gnaegi
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D004023:Dictyostelium / Q000648:ultrastructure; D055032:Electron Microscope Tomography / Q000379:methods*; D005629:Frozen Sections / Q000295:instrumentation* / Q000379:methods*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D020102:Mycobacterium smegmatis / Q000648:ultrastructure; D011328:Prions / Q000378:metabolism / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D011328:Prions