SPECIMEN PREPARATION AND QUANTIFICATION OF COLLAGEN BIREFRINGENCE IN UNSTAINED SECTIONS OF ARTICULAR-CARTILAGE USING IMAGE-ANALYSIS AND POLARIZING LIGHT-MICROSCOPY

To establish an optimal method for analysis of the collagen structures from unstained tissue sections, a computerized image analysis system using a charge coupled device camera coupled to a polarizing light mic roscope was used. Retardation values of birefringence, which are propo rtional to the content and fibril orientation of collagen in the extra cellular matrix of articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular carti lage, the highest retardation values were recorded from sections cut p arallel to the so-called split lines indicating the anisotropic arrang ement of collagen. Complete digestion of glycosaminoglycans reduced th e retardation value by approximately 6.0%, suggesting a minor, but not insignificant, contribution of glycosaminoglycans to the birefringenc e of the matrix. The use of a mounting medium with a refractive index close to that of the collagen (e.g. DPX) increased the specificity of the method, since the optical anisotropy of collagen derives predomina ntly from the intrinsic (structural) birefringence. In conclusion, ana lysis of unstained sections after careful removal of paraffin and glyc osaminoglycans from the tissues provides a sensitive and rapid quantit ative assessment of oriented collagen structures in articular cartilag e.