A synchrotron X-ray diffraction study of bovine cornea stained with cupromeronic blue

Bovine corneal stroma was stained with the dye Cupromeronic Blue under "critical electrolyte" conditions in order to locate the proteoglycans specifically. Electron microscopy revealed that most of the stained proteoglycan filaments are randomly distributed in the tissue although there are many regions where a close proteoglycan-collagen relationship is evident. On counterstaining the collagen in order to identify the intra-periodic banding pattern, the proteoglycan filaments were seen to predominate at three axial positions within the collagen D-period corresponding to electron-optical staining bands a, c and d/e. X-ray diffraction patterns were obtained from the stained and unstained corneae using a high intensity synchrotron source. Integrated intensities from the meridional patterns were used to calculate a difference electron density distribution which revealed where the collagen was axially modified by the stain. This distribution indicated the presence of the dye periodically associated with the collagen at three major sites, one adjacent to each gap/overlap junction (at electron-optical staining bands a and c) and one in the gap zone (including staining bands d and e). However, the Cupromeronic Blue treatment is estimated to have contributed only about 12% to the variation in electron density along the collagen. The staining also gave rise to a greatly increased level of diffuse background scatter, which, in agreement with the electron-optical observations, indicates that much of the stained material is not ordered with respect to the collagen but is randomly distributed in the tissue.