Averaged spacing and 2-D organization of collagen fibrils in the posterior cornea of the rabbit eye assessed by transmission electron microscopy

Purpose: To assess how reproducible collagen fibril spacing might be in the corneal stroma as viewed by transmission electron microscopy by calculating averaged values for the 2-D organization. Methods: One cornea from 8 albino rabbits (2kg) was fixed in situ to preserve natural shape. Thin sections were stained with 2% phosphotungstic acid (PTA) and images taken of fibrils from the central-posterior stroma. After projection at 250,000 X magnification, an overlay was prepared of the fibrils. Using a 500×500nm region of interest (ROI), the distances to all fibril centers were measured to 2nm resolution. Results: The sets of fibrils had average diameters between 32.4 and 36.1nm (group meanSD of 34.44.2nm). The mean fibril density was 39621 per square micrometer, with a fibril area fraction of 38.73.9%. The mean distance to the literal nearest neighbor fibril center was 43.24.5nm. A radial distribution analysis showed a distinct nearest neighbors peak at 51nm. This nearest neighbors peak had an average amplitude of 2.2360.315, with a broader secondary peak being evident in all data sets centered at 93nm with an average amplitude of 1.1660.093 (or 53.37.1% of the nearest neighbors peak). Conclusions: Overall, these results show that a predictable 2-D organization can be demonstrated for collagen fibrils in rabbit corneas when consideration is given to sample selection and preparation and the image analysis strategy.