title: Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy.
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.
authors.affiliation: Department of Anatomy, University of Kuopio, Finland.
authors.forename: K
authors.initials: K
authors.lastname: Király
authors.forename: M M
authors.initials: MM
authors.lastname: Hyttinen
authors.forename: T
authors.initials: T
authors.lastname: Lapveteläinen
authors.forename: M
authors.initials: M
authors.lastname: Elo
authors.forename: I
authors.initials: I
authors.lastname: Kiviranta
authors.forename: J
authors.initials: J
authors.lastname: Dobai
authors.forename: L
authors.initials: L
authors.lastname: Módis
authors.forename: H J
authors.initials: HJ
authors.lastname: Helminen
authors.forename: J P
authors.initials: JP
authors.lastname: Arokoski
mesh_terms: D000818:Animals; D001718:Birefringence; D002358:Cartilage, Articular / Q000737:chemistry*; D002417:Cattle; D003094:Collagen / Q000737:chemistry*; D006614:Hindlimb; D016591:Histocytological Preparation Techniques; D007091:Image Processing, Computer-Assisted; D007596:Joints; D008859:Microscopy, Polarization
chemical_list: D003094:Collagen