Helium ion microscopy for high‐resolution visualization of the articular cartilage collagen network

Abstract

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.

Postnatal changes to the mechanical properties of articular cartilage are driven by the evolution of its collagen network

“…Recently, digital image correlation techniques have been developed to characterise the depth varying compressive properties of articular cartilage (Schinagl et al, 1997;Schinagl et al, 1996;Wang et al, 2002;Canal Guterl et al, 2010). Furthermore, developments such as helium ion microscopy have enabled unprecedented nanoscale visualisation of the collagen network of articular cartilage (Vanden Berg-Foels et al, 2012), while advances in constitutive modelling are providing greater insight into how the structure and composition of articular cartilage determine the biomechanical function of the tissue (Ateshian, 2009;Korhonen et al, 2003;Nagel and Kelly, 2010;Soltz and Ateshian, 2000;Soulhat et al, 1999;Wilson et al, 2007). The objective of this study was to elucidate how the structure and composition of articular cartilage change during postnatal development and maturation using a novel combination of histological, biochemical and imaging techniques, and to relate this evolution to changes in the depth dependent mechanical properties of the tissue.…”

“…In order to provide an unobstructed view of the collagen fibrils, serial enzymatic digestion was used to remove glycosaminoglycan from the cartilage specimens; this protocol is based on a modified version of Vanden Berg-Foels et al (2012). HIM images were acquired in secondary electron mode with an acceleration voltage of 34.9 kV, a beam current of 0.2-0.5 pA and a dwell time of 0.5-2.0 µs.…”

A new structural biomarker that quantifies and predicts changes in clot strength and quality in a model of progressive haemodilution

“…More fibrils within the image can be measured because more appear in focus, an advantage already applied to imaging other biological specimens. [22][23][24] Assessment of the arrangement of the clot microstructure was achieved by qualitative assessment of the images by visual inspection focusing on the general topography of the clot, and through quantification of the fibre widths in the clot. Fibre width were measured using the computer software program ImageJ® (Rasband WS, ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA) measuring widths of 100 different fibres at each dilution, using randomly generated x,y coordinates from within multiple fields of view.…”

Ionoluminescence in the Helium Ion Microscope

“…This ensures that the primary beam remains tightly focused as it passes through the first few tens of nanometers of the sample, leading to a small interaction volume and therefore a high resolution. A probe size of 0.25 nm has been demonstrated by the microscope manufacturers (Scipioni, 2008;Vanden Berg-Foels et al, 2012). The latest HIM systems, including the one used for this work, are specified to an edge resolution, measured across the edge of a graphene flake, of <0.35 nm.…”