title: Using size-selected gold clusters on graphene oxide films to aid cryo-transmission electron tomography alignment.
abstract: A three-dimensional reconstruction of a nano-scale aqueous object can be achieved by taking a series of transmission electron micrographs tilted at different angles in vitreous ice: cryo-Transmission Electron Tomography. Presented here is a novel method of fine alignment for the tilt series. Size-selected gold clusters of ~2.7 nm (Au₅₆₁±₁₄ ), ~3.2 nm (Au₉₂₃± ₂₂ ), and ~4.3 nm (Au₂₀₅₇±₄₅) in diameter were deposited onto separate graphene oxide films overlaying holes on amorphous carbon grids. After plunge freezing and subsequent transfer to cryo-Transmission Electron Tomography, the resulting tomograms have excellent (de-)focus and alignment properties during automatic acquisition. Fine alignment is accurate when the evenly distributed 3.2 nm gold particles are used as fiducial markers, demonstrated with a reconstruction of a tobacco mosaic virus. Using a graphene oxide film means the fiducial markers are not interfering with the ice bound sample and that automated collection is consistent. The use of pre-deposited size-selected clusters means there is no aggregation and a user defined concentration. The size-selected clusters are mono-dispersed and can be produced in a wide size range including 2-5 nm in diameter. The use of size-selected clusters on a graphene oxide films represents a significant technical advance for 3D cryo-electron microscopy.
authors.affiliation: School of Biochemistry, University of Bristol, BS8 1TD, UK.
authors.forename: Kenton P
authors.initials: KP
authors.lastname: Arkill
authors.affiliation: Wolfson Bioimaging Facility, University of Bristol, BS8 1TD, UK.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Mantell
authors.affiliation: Nanoscale Physics Research Laboratory, School of Physics and Astronomy, University of Birmingham, B15 2TT, UK.
authors.forename: Simon R
authors.initials: SR
authors.lastname: Plant
authors.affiliation: 1] School of Biochemistry, University of Bristol, BS8 1TD, UK [2] Wolfson Bioimaging Facility, University of Bristol, BS8 1TD, UK [3] School of Physiology &Pharmacology, University of Bristol, BS8 1TD, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Verkade
authors.affiliation: Nanoscale Physics Research Laboratory, School of Physics and Astronomy, University of Birmingham, B15 2TT, UK.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Palmer
mesh_terms: D020285:Cryoelectron Microscopy; D055032:Electron Microscope Tomography; D006046:Gold / Q000737:chemistry*; D006108:Graphite / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry; D010087:Oxides / Q000737:chemistry; D010316:Particle Size
chemical_list: D010087:Oxides; D006046:Gold; D006108:Graphite