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.

Photonic crystal cavities from hexagonal boron nitride.

Development of scalable quantum photonic technologies requires on-chip integration of photonic components. Recently, hexagonal boron nitride (hBN) has emerged as a promising platform, following reports of hyperbolic phonon-polaritons and optically stable, ultra-bright quantum emitters. However, exploitation of hBN in scalable, on-chip nanophotonic circuits and cavity quantum electrodynamics (QED) experiments requires robust techniques for the fabrication of high-quality optical resonators. In this letter, we design and engineer suspended photonic crystal cavities from hBN and demonstrate quality (Q) factors in excess of 2000. Subsequently, we show deterministic, iterative tuning of individual cavities by direct-write EBIE without significant degradation of the Q-factor. The demonstration of tunable cavities made from hBN is an unprecedented advance in nanophotonics based on van der Waals materials. Our results and hBN processing methods open up promising avenues for solid-state systems with applications in integrated quantum photonics, polaritonics and cavity QED experiments.

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.

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.