Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy |
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Authors: | K Kiraly M M Hyttinen T Lapvetelainen M Elo I Kiviranta J Dobai L Modis H J Helminen J P A Arokoski |
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Institution: | (1) Department of Anatomy, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland;(2) Department of Surgery, Jyvaskyla Central Hospital, FIN-40620 Jyvaskyla, Finland;(3) Department of Anatomy, Histology and Embryology, The University Medical School of Debrecen, H-4012 Debrecen, Hungary;(4) Department of Physical and Rehabilitation Medicine, Kuopio University Hospital, FIN-70211 Kuopio, Finland |
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Abstract: | 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 microscope was used. Retardation values
of birefringence, which are proportional to the content and fibril orientation of collagen in the extracellular matrix of
articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular cartilage,
the highest retardation values were recorded from sections cut parallel to the so-called split lines indicating the anisotropic
arrangement of collagen. Complete digestion of glycosaminoglycans reduced the retardation value by approximately 6.0%, suggesting
a minor, but not insignificant, contribution of glycosaminoglycans to the birefringence 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 predominantly from the intrinsic (structural) birefringence. In conclusion, analysis
of unstained sections after careful removal of paraffin and glycosaminoglycans from the tissues provides a sensitive and rapid
quantitative assessment of oriented collagen structures in articular cartilage |
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