Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon

Abstract

Connective tissue polarity has remained an intractable enigma for over two decades. We present new data on optical second harmonic generation in native, wet, rat-tail tendon. Scanning second-harmonic microscopy has revealed, for the first time, the existence of a discrete network of fine, polar, filamentous or columnar, structures, and, also, the presence of strongly polar surface, or near-surface patches. The thickness of these features was probed via crossed-beam optical frequency summation and the polar material is estimated to occupy a few percent of the tendon volume. The three-dimensional spatial distribution of filaments was studied with the aid of small-angle second-harmonic scattering, and the filaments were found to permeate the tendon cross-section in an apparently random fashion. These latter measurements also revealed that essentially all polar filaments had the same directionality. Concomitant studies of the polar collagen fibrils that comprise the bulk of tendon were in full accord with prior electron microscope results that had demonstrated that the directionality of these fibrils varies up/down in a purely random fashion, and thus cannot yield a net macroscopic polarity. Quantitative analysis of the second-harmonic data yields the conclusion that the observed polar structures cannot be simply local regions containing some accidental net excess of similarly oriented fibrils. The analytical expressions used in the analysis of the data obtained for this complex tissue were supported by extensive, realistic computer simulations. The discovery that the polarity of rat-tail tendon, and possibly other forms of connective tissue, resides in discrete structures, some of which are located near the tendon surface, should permit the ready isolation of polar-rich material for further study by a variety of techniques.