Visualizing molecular polar order in tissues via electromechanical coupling

Electron microscopy (EM) and atomic force microscopy
(AFM) techniques have long been used to characterize collagen fibril
ordering and alignment in connective tissues. These techniques, however,
are unable to map collagen fibril polarity, i.e., the polar orientation
that is directed from the amine to the carboxyl termini. Using a
voltage modulated AFM-based technique called piezoresponse force
microscopy (PFM), we show it is possible to visualize both the alignment
of collagen fibrils within a tissue and the polar orientation of the
fibrils with minimal sample preparation. We demonstrate the technique on
rat tail tendon and porcine eye tissues in ambient conditions. In each
sample, fibrils are arranged into domains whereby neighboring domains
exhibit opposite polarizations, which in some cases extend to the
individual fibrillar level. Uniform polarity has not been observed in
any of the tissues studied. Evidence of anti-parallel ordering of the
amine to carboxyl polarity in bundles of fibrils or in individual
fibrils is found in all tissues, which has relevance for understanding
mechanical and biofunctional properties and the formation of connective
tissues. The technique can be applied to any biological material
containing piezoelectric biopolymers or polysaccharides.