Circularly polarized light standards for investigations of collagen fiber orientation in bone

Bone exhibits positive form birefringence dominated by and dependent upon the orientation of its collagen. The biomechanical efficacy of bone as a tissue is largely determined by collagen fibers of preferred orientation and distribution (and corresponding orientation of mineral crystallites), and evidence is accumulating to demonstrate that this efficacy extends to function at the organ level. This study has three aims. The first is to provide a Background to the study of circularly polarized light (CPL) investigations of collagen fiber orientation in bone. The significance of preferred collagen fiber orientation in bone, linearly polarized light and CPL imaging principles, and a short history of CPL studies of mammalian functional histology are reviewed. The second is to describe, in some detail, methodological considerations relating to specimen preparation and imaging appropriate for the quantitative analysis of preferentially oriented collagen. These include section transparency, section thickness, the uniformity of the illuminating system, and CPL paraphernalia. Finally, we describe a grey-level standard useful for quantitative CPL, based upon mineralized turkey tendon, which shall be provided to investigators upon request. When due consideration is paid to specimen preparation and imaging conditions, quantitative assessment of collagen fiber orientation provides insight into the effects of mechanical loading on the skeleton.