Assessment of collagen fibril spacing in relation to selected region of interest (ROI) on electron micrographs: application to the mammalian corneal stroma

The study aim was to evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center-to-center spacing) were measured within different ROIs of the same nominal area (0.25 µm2) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / µm2 and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater.