Assessment of Collagen Fibril Spacing in Relation to Selected Region of Interest (ROI) on Electron Micrographs-Application to the Mammalian Corneal Stroma

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: 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 x to obtain overlays. Interfibril distances (IFDs; center-to-center spacing) were measured within different ROIs of the same nominal area (0.25 mu m(2)) 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 / mu m(2) 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 145157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 4554 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. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech. 75:474-483, 2012. (C) 2011 Wiley Periodicals, Inc.