title: A cylindrical specimen holder for electron cryo-tomography.
abstract: The use of slab-like flat specimens for electron cryo-tomography restricts the range of viewing angles that can be used. This leads to the "missing wedge" problem, which causes artefacts and anisotropic resolution in reconstructed tomograms. Cylindrical specimens provide a way to eliminate the problem, since they allow imaging from a full range of viewing angles around the tilt axis. Such specimens have been used before for tomography of radiation-insensitive samples at room temperature, but never for frozen-hydrated specimens. Here, we demonstrate the use of thin-walled carbon tubes as specimen holders, allowing the preparation of cylindrical frozen-hydrated samples of ribosomes, liposomes and whole bacterial cells. Images acquired from these cylinders have equal quality at all viewing angles, and the accessible tilt range is restricted only by the physical limits of the microscope. Tomographic reconstructions of these specimens demonstrate that the effects of the missing wedge are substantially reduced, and could be completely eliminated if a full tilt range was used. The overall quality of these tomograms is still lower than that obtained by existing methods, but improvements are likely in future.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: cpalmer@mrc-lmb.cam.ac.uk.
authors.forename: Colin M
authors.initials: CM
authors.lastname: Palmer
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: jyl@mrc-lmb.cam.ac.uk.
authors.forename: Jan
authors.initials: J
authors.lastname: Löwe
mesh_terms: D002244:Carbon / Q000737:chemistry; D055032:Electron Microscope Tomography / Q000295:instrumentation*; D004867:Equipment Design; D004926:Escherichia coli / Q000648:ultrastructure; D008081:Liposomes / Q000648:ultrastructure; D053758:Nanoparticles / Q000737:chemistry; D012270:Ribosomes / Q000648:ultrastructure; D013048:Specimen Handling / Q000295:instrumentation* / Q000379:methods*
chemical_list: D008081:Liposomes; D002244:Carbon
keywords: Carbon nanopipettes; Cryoelectron tomography; Cylindrical specimen; Electron cryo-tomography; Missing wedge; Rod-shaped specimen