Microwave-enhanced fixation of rabbit articular cartilage

Cartilage, being highly aqueous, is difficult to preserve for electron microscopy without artefacts. Microwave-enhanced fixation is suggested as a standard method for block samples of this material, with dimensions of up to 12 × 7 × 3 mm. Cartilage samples from the tibial plateau of adult rabbits were fixed by conventional, cryo- or microwave-enhanced fixation. Constant or cyclical microwave irradiation of samples, immersed in fixatives, was carried out to varying final solution temperatures. Microwave-enhanced fixation and staining is shown to be both rapid and reproducible, giving fine structural preservation. Below 323 K microwave fixation always gave excellent preservation of the fine structure within seconds. At higher temperatures thermal artefacts were introduced. In this study the microwave-enhanced fixation is equal in quality to the best conventional immersion fixation and is nearly as fast as cryo-preservation. It provides a standardized, reproducible fixation for morphological studies on cartilage with good process control.     

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

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.     

Vitrification of articular cartilage by high-pressure freezing

For more than 20 years, high-pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high-pressure freezing. The cooling rates necessary to achieve this result under high-pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed. It was the aim of this study to explore the potential and limits of high-pressure freezing using theoretical and experimental methods. A new high-pressure freezing apparatus (Lei?a EM HPF), which can generate higher cooling rates at the sample surface than previously possible, was used. Using bovine articular cartilage as a model tissue system, we were able to vitrify 150-μm-thick tissue samples. Vitrification was proven by subjecting frozen-hydrated cryosections to electron diffraction analysis and was found to be dependent on the proteoglycan concentration and water content of the cartilage. Only the lower radical zone (with a high proteoglycan concentration and a low water content compared to the other zones) could be fully vitrified. Our theoretical calculations indicated that applied surface cooling rates in excess of 5000 K/s can be propagated into specimen centres only if samples are relatively thin (<200 μm). These calculations, taken together with our zone-dependent attainment of vitrification in 150-μm-thick cartilage samples, suggest that the critical cooling rates necessary to achieve vitrification of biological samples under high-pressure freezing conditions are significantly higher (1000–100 000 K/s) than previously proposed, but are reduced by about a factor of 100 when compared to cooling rates necessary to vitrify biological samples at ambient pressure.     

Quantification of the graphical details of collagen fibrils in transmission electron micrographs

A novel 2D image analysis technique is demonstrated. Using the digitized images of articular cartilage from transmission electron microscopy (TEM), this technique performs a localized 'vector' analysis at each region that is large enough to include several or tens of collagen fibrils but small enough to provide a fine resolution for the whole tissue. For each small and localized region, the morphology of the collagen fibrils can be characterized by three quantities essential to the nature of the tissue: the concentration of the fibrils, the overall orientation of the fibrils, and the anisotropy of the fibrils. This technique is capable of providing new insight to the existing technology by assigning quantitative attributes to the qualitative graphics. The assigned quantities are sensitive to the fine structure of the collagen matrix and meaningful in the architectural nature of the collagen matrix. These quantities could provide a critical linkage between the ultrastructure of the tissue and the macroscopic behaviours of the material. In addition, coarse-graining the microscopic resolution of EM without compromising the essential features of the tissue's structure provides a direct view of the tissue's morphology and permits direct correlations and comparisons among interdisciplinary techniques.