title: Integrative approaches for cellular cryo-electron tomography: correlative imaging and focused ion beam micromachining.
abstract: The application of cryo-electron tomography to cells and tissues is commonly referred to as "cellular tomography," and enables visualization of the supramolecular architecture of cells in a near-native state. However, in order to access structural features hidden deep inside cellular volumes, it is necessary to use hybrid techniques to identify and localize features of interest and prepare such regions for subsequent analysis by transmission electron microscopy. We present a workflow that integrates different approaches: (1) correlative cryo-fluorescence microscopy to localize features within frozen-hydrated cells, (2) focused ion beam milling to thin these specimens in a targeted manner, and (3) cryo-electron tomography to provide detailed information about the cellular ultrastructure of thinned samples. We describe the combined use of these techniques and the instrumentation required to enable cryo-electron tomography for a vast range of cellular samples.
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: Elizabeth
authors.initials: E
authors.lastname: Villa
authors.forename: Felix J B
authors.initials: FJ
authors.lastname: Bäuerlein
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Engel
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy; D015925:Cryopreservation; D004023:Dictyostelium / Q000648:ultrastructure; D055032:Electron Microscope Tomography; D005629:Frozen Sections; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D042282:Microdissection; D008856:Microscopy, Fluorescence; D020102:Mycobacterium smegmatis / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000648:ultrastructure; D058989:Vitrification
chemical_list: D049452:Green Fluorescent Proteins