title: Cryo-electron microscopy specimen preparation by means of a focused ion beam.
abstract: Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.
authors.affiliation: Department of Engineering Sciences, Uppsala University; Physics Department, University of Oslo; stefanorubino@yahoo.it.
authors.forename: Stefano
authors.initials: S
authors.lastname: Rubino
authors.affiliation: Department of Microbiology, Swedish University of Agricultural Sciences.
authors.forename: Petter
authors.initials: P
authors.lastname: Melin
authors.affiliation: Gatan Inc.
authors.forename: Paul
authors.initials: P
authors.lastname: Spellward
authors.affiliation: Department of Engineering Sciences, Uppsala University; klaus.leifer@angstrom.uu.se.
authors.forename: Klaus
authors.initials: K
authors.lastname: Leifer
mesh_terms: D001234:Aspergillus niger / Q000648:ultrastructure; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D007477:Ions / Q000737:chemistry; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation / Q000379:methods; D013172:Spores, Fungal / Q000648:ultrastructure
chemical_list: D007477:Ions