Sample preparation is a critical step in scanning electron microscopy (SEM) imaging. This is especially true for biological samples because of charge build-up and sensitivity to vacuum and electron beam damage. In terms of ultrastructure imaging, a variety of advancements in detectors and approaches have improved biological imaging such that fewer steps are required for sample preparation. However, the conventional approach incorporating osmium tetroxide fixing, ethanol dehydrating, critical-point drying, and coating still finds useful application. This paper evaluates three biological sample-preparation methodologies for imaging the ultrastructure of immature porcine retina. The three preparation methods examined are critical-point drying (CPD), hexamethyldisilazane (HMDS) dehydration, and direct imaging by environmental scanning electron microscopy (ESEM). Preparation methodologies were evaluated based on resulting image quality and reduced potential for artifacts.

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An advanced rheological technique was used to estimate the dynamic material properties of pediatric porcine vitreous. Validation of the technique was performed with two simulant materials. Interconversion of time-dependent data to frequency-dependent data resulted in good approximation of storage and loss modulus for a primarily viscous material, but resulted in an erroneous loss modulus for a primarily elastic material. This can likely be overcome by utilizing free oscillations resulting from creep testing. Porcine storage modulus was significantly lower in older animals, but additional testing is necessary to completely characterize the material properties of pediatric vitreous over a wide range of frequencies.