Options for determination of 2-D distribution of collagen fibrils in transmission electron micrographs: application to the mammalian corneal stroma

The aim of this study was to assess methods for both measurement of and comparison between fibril spacing arrangement in transmission electron micrograph (TEM) images of collagen fibrils. Rabbit corneas were glutaraldhyde fixed, treated with osmium tetroxide and then thin sections stained with aqueous phosphotungstic acid. TEM images, at 28,000 magnification, of collagen fibrils in cross section were projected at a final magnification close to 250,000 to obtain overlays. Fibril centers were marked and the relative position of all fibrils measured either from a cluster of fibrils within a fixed region of interest (ROI) or by using a variable sequential ROI across the micrograph. The 2-D organization of the interfibril distance (IFD) data were analyzed using a radial distribution analysis and the difference in outcome statistically compared.