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Cryo-FIB specimen preparation for use in a cartridge-type cryo-TEM. 2017

Jie He, and Chyongere Hsieh, and Yongping Wu, and Thomas Schmelzer, and Pan Wang, and Ying Lin, and Michael Marko, and Haixin Sui
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201, USA.

Cryo-electron tomography (cryo-ET) is a well-established technique for studying 3D structural details of subcellular macromolecular complexes and organelles in their nearly native context in the cell. A primary limitation of the application of cryo-ET is the accessible specimen thickness, which is less than the diameters of almost all eukaryotic cells. It has been shown that focused ion beam (FIB) milling can be used to prepare thin, distortion-free lamellae of frozen biological material for high-resolution cryo-ET. Commercial cryosystems are available for cryo-FIB specimen preparation, however re-engineering and additional fixtures are often essential for reliable results with a particular cryo-FIB and cryo-transmission electron microscope (cryo-TEM). Here, we describe our optimized protocol and modified instrumentation for cryo-FIB milling to produce thin lamellae and subsequent damage-free cryotransfer of the lamellae into our cartridge-type cryo-TEM.

UI MeSH Term Description Entries
D008722 Methods A series of steps taken in order to conduct research. Techniques,Methodological Studies,Methodological Study,Procedures,Studies, Methodological,Study, Methodological,Method,Procedure,Technique
D004867 Equipment Design Methods and patterns of fabricating machines and related hardware. Design, Equipment,Device Design,Medical Device Design,Design, Medical Device,Designs, Medical Device,Device Design, Medical,Device Designs, Medical,Medical Device Designs,Design, Device,Designs, Device,Designs, Equipment,Device Designs,Equipment Designs
D005629 Frozen Sections Thinly cut sections of frozen tissue specimens prepared with a cryostat or freezing microtome. Frozen Section,Section, Frozen,Sections, Frozen
D013048 Specimen Handling Procedures for collecting, preserving, and transporting of specimens sufficiently stable to provide accurate and precise results suitable for clinical interpretation. Specimen Collection,Collection, Specimen,Collections, Specimen,Handling, Specimen,Handlings, Specimen,Specimen Collections,Specimen Handlings
D015003 Yeasts A general term for single-celled rounded fungi that reproduce by budding. Brewers' and bakers' yeasts are SACCHAROMYCES CEREVISIAE; therapeutic dried yeast is YEAST, DRIED. Yeast
D046529 Microscopy, Electron, Transmission Electron microscopy in which the ELECTRONS or their reaction products that pass down through the specimen are imaged below the plane of the specimen. Electron Diffraction Microscopy,Electron Microscopy, Transmission,Microscopy, Electron Diffraction,Transmission Electron Microscopy,Diffraction Microscopy, Electron,Microscopy, Transmission Electron
D020285 Cryoelectron Microscopy Electron microscopy involving rapid freezing of the samples. The imaging of frozen-hydrated molecules and organelles permits the best possible resolution closest to the living state, free of chemical fixatives or stains. Electron Cryomicroscopy,Cryo-electron Microscopy,Cryo electron Microscopy,Cryo-electron Microscopies,Cryoelectron Microscopies,Cryomicroscopies, Electron,Cryomicroscopy, Electron,Electron Cryomicroscopies,Microscopies, Cryo-electron,Microscopies, Cryoelectron,Microscopy, Cryo-electron,Microscopy, Cryoelectron

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