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.