Assessment of the apparent intra-and inter-sample variability in collagen fibril diameter in the posterior corneal stroma of rabbits: a transmission electron microscopy study

Purpose: To assess the natural variability in the diameter of small collagen fibrils in the lamellae of corneal stroma of healthy young rabbits. Materials and Methods: The corneas of 6 young adult female New Zealand White rabbits (2.1 kg) were prepared for transmission electron microscopy (TEM) by fixation with a cacodylate-buffered 2% glutaraldehyde (pH 7.2–7.4, 320–340 mosm/kg). The corneas were embedded in Epon-Araldite, thin sections prepared from the central region of the posterior corneal stroma and stained with acidic uranyl acetate followed by acidic lead citrate. High magnification (×33,000) micrographs were taken, and fibril diameters (FDs) measured at a final magnification of ×275,000 to a resolution of 2 nm. Results: Assessment of sampling-related errors indicated that the average diameter of the fibrils within any particular micrograph could be estimated to within 1% or better by measures of 100 fibrils. Assessments of the intra-sample variance (6 micrographs taken from the same cornea) indicated a group mean FD of 32.4 ± 3.6 nm, whilst the inter-sample variance (6 micrographs taken from 6 different corneas) yielded an average of 33.1 ± 4.5 nm (n = 100 fibrils/micrograph, ±SD). However, group-averaged data sets of FDs, while unimodal, were not normally distributed, and cumulative averaging indicated a fixed range of FDs across the data sets. Conclusions: Intra- and inter-sample variability in collagen FDs is very similar, but the analysis indicates that the collagen fibrils are not homogeneous and that closely adjacent lamellae can have subtle differences in average FD.