Optical antenna structures have revolutionized the field of nano-optics by confining light to deep subwavelength dimensions for spectroscopy and sensing. In this work, we fabricated coaxial optical antennae with sub-10-nanometer critical dimensions using helium ion lithography (HIL). Wavelength dependent transmission measurements were used to determine the wavelength-dependent optical response. The quality factor of 11 achieved with our HIL fabricated structures matched the theoretically predicted quality factor for the idealized flawless gold resonators calculated by finite-difference time-domain (FDTD). For comparison, coaxial antennae with 30 nm critical dimensions were fabricated using both HIL and the more common Ga focus ion beam lithography (Ga-FIB). The quality factor of the Ga-FIB resonators was 60% of the ideal HIL results for the same design geometry due to limitations in the Ga-FIB fabrication process.

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.

Articles you may be interested inAnalysis of subsurface beam spread and its impact on the image resolution of the helium ion microscope Recent investigations are gaining us a better understanding of the nature of the beam-sample interactions in the helium ion microscope and what they mean for the image information provided. In secondary electron ͑SE͒ imaging, for example, the surface sensitivity is attributed to the low SE-II fraction. Voltage contrast imaging shows the ability to see both buried structures and to probe the conductance to ground of surface contacts. It is found, however, that the prominence of these two types of contrast varies oppositely with beam energy, yielding information about the nature of the interactions that gives rise to them. Transmission ion imaging can yield information about material density, atomic number, grain structure, and electronic structure. It is possible to capture the top-side SE signal, bright field signal, and dark field signal from a given sample simultaneously. The detection of diffraction contrast is under investigation.

The articular cartilage collagen network is an important research focus because network disruption results in cartilage degeneration and patient disability. The recently introduced helium ion microscope (HIM), with its smaller probe size, longer depth of field and charge neutralization, has the potential to overcome the inherent limitations of electron microscopy for visualization of collagen network features, particularly at the nanoscale. In this study, we evaluated the capabilities of the helium ion microscope for high-resolution visualization of the articular cartilage collagen network. Images of rabbit knee cartilage were acquired with a helium ion microscope; comparison images were acquired with a field emission scanning electron microscope (FE-SEM) and a transmission electron microscope (TEM). Sharpness of example high-resolution helium ion microscope and field emission scanning electron microscope images was quantified using the 25-75% rise distance metric. The helium ion microscope was able to acquire high-resolution images with unprecedented clarity, with greater sharpness and three-dimensional-like detail of nanoscale fibril morphologies and fibril connections, in samples without conductive coatings. These nanoscale features could not be resolved by field emission scanning electron microscopy, and three-dimensional network structure could not be visualized with transmission electron microscopy. The nanoscale three-dimensional-like visualization capabilities of the helium ion microscope will enable new avenues of investigation in cartilage collagen network research.

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