Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas phase and solution phase protein structures.
Gas-mediated electron beam induced etching (EBIE) and deposition (EBID) can be used to measure activation energies that are interpreted as the adsorption energies of surface-adsorbed precursor molecules. However, the measured quantities often disagree with adsorption energies measured by conventional analysis techniques such as thermally programmed desorption and have anomalous dependencies on parameters such as the electron beam current used to perform EBID. Here, we use the theory of EBIE and EBID rate kinetics to explain this behavior and identify conditions under which the activation energies and the associated pre-exponential factors correspond to gas molecule adsorption energies and desorption attempt frequencies, respectively. Under these conditions, EBIE and EBID can be used as robust, nanoscale techniques for the analysis of adsorbates.
Electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA) have revolutionized structure determination of homogeneous proteins. However, obtaining high-resolution structures from heterogeneous samples remains a major challenge, as the various protein states embedded in thin films of vitreous ice may be classified incorrectly, resulting in detrimental averaging of features. Here we present native electrospray ion-beam deposition (native ES-IBD) for the preparation of extremely high-purity cryo-EM samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, and subsequently frozen in liquid nitrogen. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected proteins and protein assemblies. SPA reveals that they remain structurally intact, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM structure determination.
Modifying material properties at the nanoscale is crucially important for devices in nano-electronics, nanophotonics and quantum information. Optically active defects in wide band gap materials, for instance, are critical constituents for the realisation of quantum technologies. Here, we demonstrate the use of recoil implantation, a method exploiting momentum transfer from accelerated ions, for versatile and mask-free material doping. As a proof of concept, we direct-write arrays of optically active defects into diamond via momentum transfer from a Xe+ focused ion beam (FIB) to thin films of the group IV dopants pre-deposited onto a diamond surface. We further demonstrate the flexibility of the technique, by implanting rare earth ions into the core of a single mode fibre. We conclusively show that the presented technique yields ultra-shallow dopant profiles localised to the top few nanometres of the target surface, and use it to achieve sub-50 nm positional accuracy. The method is applicable to non-planar substrates with complex geometries, and it is suitable for applications such as electronic and magnetic doping of atomically-thin materials and engineering of near-surface states of semiconductor devices.
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