title: Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography.
abstract: Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells - especially those in tissue - are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.
authors.affiliation: New York State Department of Health, Wadsworth Center, Empire State Plaza, Albany, NY 12201, USA.
authors.forename: Chyongere
authors.initials: C
authors.lastname: Hsieh
authors.affiliation: TGS Technologies, 702 Little Creek Lane, Cranberry Township, PA 16066, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmelzer
authors.affiliation: New York State Department of Health, Wadsworth Center, Empire State Plaza, Albany, NY 12201, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Kishchenko
authors.affiliation: New York State Department of Health, Wadsworth Center, Empire State Plaza, Albany, NY 12201, USA; Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12203, USA.
authors.forename: Terence
authors.initials: T
authors.lastname: Wagenknecht
authors.affiliation: New York State Department of Health, Wadsworth Center, Empire State Plaza, Albany, NY 12201, USA; College of Nanoscale Science and Engineering, University at Albany, 251 Fuller Rd., Albany, NY 12203, USA. Electronic address: marko@wadsworth.org.
authors.forename: Michael
authors.initials: M
authors.lastname: Marko
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D055032:Electron Microscope Tomography / Q000295:instrumentation* / Q000379:methods*; D005615:Freezing; D005629:Frozen Sections / Q000295:instrumentation / Q000379:methods; D007477:Ions / Q000737:chemistry; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation* / Q000379:methods*; D008867:Microtomy / Q000295:instrumentation / Q000379:methods; D057188:Workflow
chemical_list: D007477:Ions
keywords: Cryo-FIB; Cryo-SEM; Cryo-TEM; Cryo-tomography; Vitreous sections