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.
Highlights
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
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
By using size-selected gold clusters pre-deposited on an ultra-thin support film, we present here a novel way of acquiring cryo-TEM tomograms using Tobacco Mosaic Virus (TMV) as an example
Summary
Prepared (Agar Scientific) TEM grids were sourced as a substrate. These arrive covered with a graphene oxide (GO) film supported on top of a perforated carbon film (Quantifoil). The TEM grids were glow discharged (EDWARDS AUTO 306) to aide wetting This had no observable effect on the gold clusters. 4 ml of TMV in aqueous media was placed on top of the grid and left for 10 s before blotting (1 s) and plunging This method resulted in a set up where the gold clusters are on one side of the GO layer whereas the vitreous ice is suspended inside the holes of the carbon film on the other side of the GO film (Figure 1). The electron flux at 0 degrees was measured as 2.6 e.A 22.s21 ( 68 e.A 22.s21 total) using the measured current on the phosphorescent screen, i.e. low dose conditions were properly observed
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