Recent evidence supports the notion that biological functions of
extracellular matrix (ECM) are highly correlated to not only its
composition but also its structure. This article integrates confocal
microscopy imaging and image-processing techniques to analyze the
microstructural properties of ECM. This report describes a two- and
three-dimensional fiber middle-line tracing algorithm that may be used
to quantify collagen fibril organization. We utilized computer
simulation and statistical analysis to validate the developed
algorithm. These algorithms were applied to confocal images of collagen
gels made with reconstituted bovine collagen type I, to demonstrate the
computation of orientations of individual fibers.