Options for determination of 2‐D distribution of collagen fibrils in transmission electron micrographs—Application to the mammalian corneal stroma

Abstract

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. Repeated measures of IFDs provides data on the nearest neighbor distance within a fixed or sequential ROI of close to 48 nm and provides a numerical descriptor of the radial positions of all other fibril centers. From the set of radial positions (IFD) data, simple statistical comparisons can be made to both assess location-to-location and sample-to-sample variability. The sequential use of an ROI of fibril arrays however provide a more complete and accurate numerical descriptor. Methods are presented that will allow for assessment of the nearest neighbors distances and the overall 2-D pattern of collagen fibrils in electron micrographs. The sets of IFDs and their pattern can be compared using simple statistical analyses.