Király K, Hyttinen M M, Lapveteläinen T, Elo M, Kiviranta I, Dobai J, Módis L, Helminen H J, Arokoski J P
Department of Anatomy, University of Kuopio, Finland.
Histochem J. 1997 Apr;29(4):317-27. doi: 10.1023/a:1020802631968.
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.
为建立一种分析未染色组织切片中胶原结构的最佳方法，使用了一种计算机图像分析系统，该系统将电荷耦合器件相机与偏光显微镜相连。从以不同方式制备的切片中测定双折射的延迟值，该值与关节软骨细胞外基质中胶原的含量和纤维取向成正比。在关节软骨的表层区域，平行于指示胶原各向异性排列的所谓分裂线切割的切片记录到最高的延迟值。糖胺聚糖的完全消化使延迟值降低了约6.0%，表明糖胺聚糖对基质双折射有微小但并非微不足道的贡献。使用折射率接近胶原的封片剂（如DPX）提高了该方法的特异性，因为胶原的光学各向异性主要源于固有（结构）双折射。总之，在仔细去除组织中的石蜡和糖胺聚糖后对未染色切片进行分析，可对关节软骨中定向胶原结构进行灵敏且快速的定量评估。
