Averaged Spacing and 2-D Organization of Collagen Fibrils in the Posterior Cornea of the Rabbit Eye Assessed by Transmission Electron Microscopy

Abstract

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 (2 kg) 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 × 500 nm region of interest (ROI), the distances to all fibril centers were measured to 2 nm resolution.

Results: The sets of fibrils had average diameters between 32.4 and 36.1 nm (group mean ± SD of 34.4 ± 4.2 nm). The mean fibril density was 396 ± 21 per square micrometer, with a fibril area fraction of 38.7 ± 3.9%. The mean distance to the literal nearest neighbor fibril center was 43.2 ± 4.5 nm. A radial distribution analysis showed a distinct nearest neighbors peak at 51 nm. This nearest neighbors peak had an average amplitude of 2.236 ± 0.315, with a broader secondary peak being evident in all data sets centered at 93 nm with an average amplitude of 1.166 ± 0.093 (or 53.3 ± 7.1% 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.

• Collagen fibrils
• image analysis
• inter-fibril spacing
• nearest neighbors distance
• radial distribution
• transmission electron microscopy