Cryo-EM of soft-landed ß-galactosidase: Gas-phase and native structures are remarkably similar.

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of ß-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM.

Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells.

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

Nanoscale focused electron beam induced etching of nickel using a liquid reactant.

Nickel nanostructures have found widespread application as both functional components, e.g. in magnetic systems, and as part of the lithographic pattern transfer process as etch masks, EUV mask absorbers, and imprint templates. Electron-beam induced etching of nickel is highly desirable for the repair and editing of masks and templates with high resolution and without substrate damage. However, there are no known gas-phase reactants that produce volatile nickel products under e-beam irradiation. Here we report the successful local etching of nickel by a focused electron beam in an environmental scanning electron microscope using a liquid reactant, aqueous sulfuric acid. Sulfuric acid did not spontaneously etch nickel under ESEM conditions, but nickel was etched in areas exposed to the electron beam. Etching parameters such as dose, refresh time, and addition of a surfactant were investigated. The extent of the etch increases with dose before terminating at sub-micron feature sizes. The etch resolution improves with the addition of surfactant. This approach enables local nickel patterning with complete film removal but without damaging underlying layers. With further refinement, the process may enable nickel absorber repair and editing and remove a significant obstacle to the use of nickel in EUV lithography.

Radiation-Induced Damage and Recovery of Ultra-Nanocrystalline Diamond: Toward Applications in Harsh Environments.

Ultra-nanocrystalline diamond (UNCD) is increasingly being used in the fabrication of devices and coatings due to its excellent tribological properties, corrosion resistance, and biocompatibility. Here, we study its response to irradiation with kiloelectronvolt electrons as a controlled model for extreme ionizing environments. Real time Raman spectroscopy reveals that the radiation-damage mechanism entails dehydrogenation of UNCD grain boundaries, and we show that the damage can be recovered by annealing at 883 K. Our results have significant practical implications for the implementation of UNCD in extreme environment applications, and indicate that the films can be used as radiation sensors.

Focused electron beam induced etching of copper in sulfuric acid solutions.

We show here that copper can be locally etched by an electron-beam induced reaction in a liquid. Aqueous sulfuric acid (H2SO4) is utilized as the etchant and all experiments are conducted in an environmental scanning electron microscope. The extent of etch increases with liquid thickness and dose, and etch resolution improves with H2SO4 concentration. This approach shows the feasibility of liquid phase etching for material selectivity and has the potential for circuit editing.

Maskless milling of diamond by a focused oxygen ion beam.

Recent advances in focused ion beam technology have enabled high-resolution, maskless nanofabrication using light ions. Studies with light ions to date have, however, focused on milling of materials where sub-surface ion beam damage does not inhibit device performance. Here we report on maskless milling of single crystal diamond using a focused beam of oxygen ions. Material quality is assessed by Raman and luminescence analysis, and reveals that the damage layer generated by oxygen ions can be removed by non-intrusive post-processing methods such as localised electron beam induced chemical etching.

Liquid phase electron-beam-induced deposition on bulk substrates using environmental scanning electron microscopy.

The introduction of gases, such as water vapor, into an environmental scanning electron microscope is common practice to assist in the imaging of insulating or biological materials. However, this capability may also be exploited to introduce, or form, liquid phase precursors for electron-beam-induced deposition. In this work, the authors report the deposition of silver (Ag) and copper (Cu) structures using two different cell-less in situ deposition methods--the first involving the in situ hydration of solid precursors and the second involving the insertion of liquid droplets using a capillary style liquid injection system. Critically, the inclusion of surfactants is shown to drastically improve pattern replication without diminishing the purity of the metal deposits. Surfactants are estimated to reduce the droplet contact angle to below ~10°.

Spontaneous growth of gallium-filled microcapillaries on ion-bombarded GaN.

Bottom-up growth of microscopic pillars is observed at room temperature on GaN irradiated with a Ga+ beam in a gaseous XeF2 environment. Ion bombardment produces Ga droplets which evolve into pillars, each comprised of a spherical Ga cap atop a Ga-filled, gallium fluoride tapered tube (sheath). The structures form through an interdependent, self-ordering cycle of liquid cap growth and solid sheath formation. The sheath and core growth mechanisms are not catalytic, but instead consistent with a model of ion-induced Ga and F generation, Ga transport through surface diffusion, and heterogeneous sputtering caused by self-masking of the tapered pillars.

Focused electron-beam-induced deposition of 3 nm dots in a scanning electron microscope.

Electron-beam-induced deposition allows the creation of three-dimensional nanodevices within a scanning electron microscope. Typically the dimensions of the fabricated structure are from 20 nm to several micrometers. Until now the record for the smallest deposited feature in an SEM was 3.5 nm, measured by an indirect method. We have achieved a nanodot having a full width half-maximum of 2.8 +/- 0.3 nm, measured directly in the same microscope after deposition.