Image analysis of tendon helical superstructure using interference and polarized light microscopy.

Wave-like structures (WLS also known as crimp) have generally been reported to be planar structures. However, there is evidence that a helical superstructure, rather than a planar one, should be considered. Conditions dictated by supramolecular chemistry, molecular recognition and self-assembly favor the idea of a helical arrangement for collagen bundles in a supramolecular structure. The aim of this work is to provide additional data in support of a helical superstructure for collagen bundles in tendons. Cryosections of fixed flexor bovine tendons and sections of resin-embedded peeled rat tail were studied using polarized light, interference, and phase contrast microscopy. Image analysis was used to find appropriate mathematical descriptors for WLS. Interference colors due to the dispersion of birefringence allowed the detection of a gradual, intertwined twisted fiber organization in WLS, as the angle of the tendon axis was rotated relative to the polarizers. Helical movements of the images of the WLS bands were produced using animation methods. Interference microscopy revealed interference colors associated with different orientations and dry mass concentrations in the fibers, especially in tendon cross-sections, which also exhibited Maltese-cross birefringence images. Similar images were detected by interference microscopy, suggesting a spiral organization of fibers in the section plane. The helical orientation of the fibers was detected by focusing through different planes of sections. Based on a comparison of this superstructure with mesophases, the twisted grain boundary concept is considered to be the most appropriate for the classification of tendon WLS.