Chuong Huynh

Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.

Nano Josephson superconducting tunnel junctions in YBa2Cu3O7–δ directly patterned with a focused helium ion beam

Since the discovery of the high-transition-temperature superconductors (HTSs), researchers have explored many methods to fabricate superconducting tunnel junctions from these materials for basic science purposes and applications. HTS circuits operating at liquid-nitrogen temperatures (∼77 K) would significantly reduce power requirements in comparison with those fabricated from conventional superconductors. The difficulty is that the superconducting coherence length is very short and anisotropic in these materials, typically ∼2 nm in the a-b plane and ∼0.2 nm along the c axis. The electrical properties of Josephson junctions are therefore sensitive to chemical variations and structural defects on atomic length scales. To make multiple uniform HTS junctions, control at the atomic level is required. In this Letter we demonstrate all-HTS Josephson superconducting tunnel junctions created by using a 500-pm-diameter focused beam of helium ions to directly write tunnel barriers into YBa2Cu3O(7-δ) (YBCO) thin films. We demonstrate the ability to control the barrier properties continuously from conducting to insulating by varying the irradiation dose. This technique could provide a reliable and reproducible pathway for scaling up quantum-mechanical circuits operating at liquid-nitrogen temperatures, as well as an avenue to conduct novel planar superconducting tunnelling studies for basic science.

Analysis and metrology with a focused helium ion beama)

The newly introduced ORION™ helium ion microscope has been used for high resolution imaging and nanofabrication. More recently, an energy sensitive detector has been developed that permits the measurement of the energy spectrum of the backscattered helium ions. The spectra can be analyzed directly or compared with the simulated spectra from hypothetical models of the specimen. The technique can provide information about the elemental composition of the specimen or structural information (for example, layer thickness) of the specimen.

High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa

We examined the rat and mouse epididymis using helium ion microscopy (HIM), a novel imaging technology that uses a scanning beam of He(+) ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning and dehydration methods were evaluated for their ability to preserve tissue architecture. The cauda epididymidis was luminally perfused in vivo to remove most spermatozoa and the apical surface of the epithelial lining was exposed. Fixed epididymis samples were then subjected to critical point drying (CPD) and HIM. Apical stereocilia in principal cells and smaller apical membrane extensions in clear cells were clearly distinguishable in both rat and mouse epididymis using this technology. After perfusion with an activating solution containing CPT-cAMP, a permeant analog of cAMP, clear cells exhibited an increase in the number and size of membrane ruffles or microplicae. In contrast, principal cells did not exhibit detectable structural modifications. High-resolution HIM imaging clearly showed the ultrastructure of residual sperm cells, including the presence of concentric rings on the midpiece, and of cytoplasmic droplets in some spermatozoa. Close epithelium-sperm interactions were also detected. We found a number of sperm cells whose heads were anchored within the epididymal epithelium. In certain cases, the surface of the sperm cytoplasmic droplet was covered with vesicle-like structures whose size is consistent with that of epididymosomes. In conclusion, we describe here the first application of HIM technology to the study of the structure and morphology of the rodent epididymis. HIM technology represents a major imaging breakthrough that can be successfully applied to study the epididymis and spermatozoa, with the goal of advancing our understanding of their structure and function.

YBa2Cu3O7−δ superconducting quantum interference devices with metallic to insulating barriers written with a focused helium ion beam

In this work, we demonstrate the ability to fabricate superconducting quantum interference devices (SQUIDs) by directly writing Josephson junctions into the plane of YBa2Cu3O7−δ thin films with a focused helium ion beam. This technique allows for the control of the Josephson barrier transport properties through the single parameter, ion dose. SQUIDs written with a dose of 4 × 1016 ions/cm2 had metallic barrier junctions that exhibited nearly ideal electrical transport characteristics at 50 K and a flux noise of 20 μΦ0/Hz at 10 Hz. At higher irradiation doses, the SQUIDs had insulating barrier Josephson junctions with a quasi particle energy gap edge at 20 meV.

Creating nanohole arrays with the helium ion microscope

Helium Ion Microscopy has been established as a powerful imaging technique offering unique contrast and high resolution surface information. More recently, the helium ion beam has been used for nanostructuring applications similar to a gallium focused ion beam. A key difference between helium and gallium induced sputtering is the less intense damage cascade which lends this technique to precise and controlled milling of different materials enabling applications. The helium ion beam has been used for drilling 5nm holes in a 100nm gold foil (20:1 aspect ratio) while the gallium beam sputtered holes of a similar aspect ratio seem to be limited to a 50nm hole size. This paper explores the drilling of nanopores in gold films and other materials and offers an explanation for the observed differences in results between helium and gallium ions.

Helium ion microscopy of enamel crystallites and extracellular tooth enamel matrix

An unresolved problem in tooth enamel studies has been to analyze simultaneously and with sufficient spatial resolution both mineral and organic phases in their three dimensional (3D) organization in a given specimen. This study aims to address this need using high-resolution imaging to analyze the 3D structural organization of the enamel matrix, especially amelogenin, in relation to forming enamel crystals. Chemically fixed hemi-mandibles from wild type mice were embedded in LR White acrylic resin, polished and briefly etched to expose the organic matrix in developing tooth enamel. Full-length amelogenin was labeled with specific antibodies and 10 nm immuno-gold. This allowed us to use and compare two different high-resolution imaging techniques for the analysis of uncoated samples. Helium ion microscopy (HIM) was applied to study the spatial organization of organic and mineral structures, while field emission scanning electron microscopy (FE-SEM) in various modes, including backscattered electron detection, allowed us to discern the gold-labeled proteins. Wild type enamel in late secretory to early maturation stage reveals adjacent to ameloblasts a lengthwise parallel alignment of the enamel matrix proteins, including full-length amelogenin proteins, which then transitions into a more heterogeneous appearance with increasing distance from the mineralization front. The matrix adjacent to crystal bundles forms a smooth and lacey sheath, whereas between enamel prisms it is organized into spherical components that are interspersed with rod-shaped protein. These findings highlight first, that the heterogeneous organization of the enamel matrix can be visualized in mineralized en bloc samples. Second, our results illustrate that the combination of these techniques is a powerful approach to elucidate the 3D structural organization of organic matrix molecules in mineralizing tissue in nanometer resolution.

Large Photothermal Effect in Sub‐40 nm h‐BN Nanostructures Patterned Via High‐Resolution Ion Beam

The controlled nanoscale patterning of 2D materials is a promising approach for engineering the optoelectronic, thermal, and mechanical properties of these materials to achieve novel functionalities and devices. Herein, high-resolution patterning of hexagonal boron nitride (h-BN) is demonstrated via both helium and neon ion beams and an optimal dosage range for both ions that serve as a baseline for insulating 2D materials is identified. Through this nanofabrication approach, a grating with a 35 nm pitch, individual structure sizes down to 20 nm, and additional nanostructures created by patterning crystal step edges are demonstrated. Raman spectroscopy is used to study the defects induced by the ion beam patterning and is correlated to scanning probe microscopy. Photothermal and scanning near-field optical microscopy measure the resulting near-field absorption and scattering of the nanostructures. These measurements reveal a large photothermal expansion of nanostructured h-BN that is dependent on the height to width aspect ratio of the nanostructures. This effect is attributed to the large anisotropy of the thermal expansion coefficients of h-BN and the nanostructuring implemented. The photothermal expansion should be present in other van der Waals materials with large anisotropy and can lead to applications such as nanomechanical switches driven by light.

Advanced Nanofabrication using Helium, Neon and Gallium Ion Beams in the Carl Zeiss Orion NanoFab Microscope

Direct write focused ion beam (FIB) machining represents the fastest and most flexible method to fabricate nano-scaled devices for prototyping and research applications. The use of a FIB combined with SEM allows for immediate inspection and refinement steps in the patterning process to assure the desired fidelity. FIB technology has thus found wide-spread use in fields such as photonics, nanofluidics, TEM sample preparation, integrated circuit modification, MEMS and more. Conventional gallium FIBs use a liquid metal ion source (LMIS) with many notable drawbacks such as a lower limit for feature sizes that can be achieved and undesirable Ga implantation. Ion microscopy with helium and neon beams created from a gas field ion source (GFIS) demonstrates great flexibility for many nanofabrication applications. The beam-sample interaction dynamics of helium and neon beams at moderate voltages extends direct write fabrication and inspection further into the sub-10 nm regime. The Carl Zeiss Orion NanoFab is a commercially available ion beam microscope offering three different species of ions to extend beyond the limits of gallium with the addition of helium and neon beams.