Depth-profiled synchrotron microbeam analyses showed that, at different radial positions through the corneal centre outwards, fibril size was higher than in deeper stromal locations superficially. the current presence of both high- and low-sulphated epitopes of KS, aswell as DS, through the entire cornea, and CS just in the peripheral cornea prior to the limbus. Quantification by ELISA, disclosed that although both high- and low-sulphated KS continued to be constant throughout stromal depth at different radial positions, high-sulphated epitopes remained constant from the corneal centre to outer-periphery, whereas low-sulphated epitopes increased significantly. Both small angle X-ray diffraction and TEM analysis revealed that collagen fibril diameter remained relatively constant until the outer periphery was reached, after which fibrils became more widely spaced (from small angle x-ray diffraction analysis) and of larger diameter as they approached the sclera. Depth-profiled synchrotron microbeam analyses showed that, at different radial positions from the corneal centre outwards, fibril diameter was greater superficially than in deeper stromal regions. The interfibrillar spacing was also MAC glucuronide α-hydroxy lactone-linked SN-38 higher at mid-depth MAC glucuronide α-hydroxy lactone-linked SN-38 in the stroma than it was in anterior and posterior stromal regions. Collagen fibrils in the bovine cornea exhibited a fairly consistent spacing and diameter from the corneal centre to the 12?mm radial position, after which a significant increase was seen. While the constancy of the overall sulphation levels of proteoglycans in the cornea may correlate with the fibrillar architecture, there was no correlation between the latter and the KIAA0243 distribution of low-sulphated KS. MAC glucuronide α-hydroxy lactone-linked SN-38 in 0.5% uranyl acetate and subsequently dehydrated through an ascending ethanol series. After transferring the samples to propylene oxide, the samples were infiltrated and embedded in Araldite CY212 resin. Ultrathin sections (90C100?nm thick, cut with diamond knife) were collected on uncoated G300 copper grids, and stained with 1% aqueous phosphotungstic acid and uranyl acetate. Sections were examined in a transmission electron microscope (JEOL 1010; JEOL, Tokyo, Japan) equipped with a charge-coupled device camera (Orius SC1000; Gatan, Pleasanton, CA). 4.9. Small-angle X-ray diffraction Fresh, clear bovine corneas were excised with a scleral rim, approximately 2C3? mm wide still attached, carefully wrapped in Clingfilm (Tesco, UK) to minimize dehydration, frozen to ??80?C and transported on dry ice to the Spring-8 synchrotron facility in Hyogo Prefecture, Japan for X-ray fibre diffraction analysis. On the high-flux beamline 40XU, the corneas were gently thawed and a 5? mm strip was cut horizontally across the central optical axis. The corneal strip was immediately placed between a single layer of Clingfilm to limit dehydration during exposure to the X-ray beam. The corneal strip was then mounted in the path of the beam that was focused to a diameter of 25?m at the anterior surface of the cornea with the epithelium facing the X-ray beam. X-ray diffraction patterns MAC glucuronide α-hydroxy lactone-linked SN-38 were obtained in 100?m steps from the corneal centre to its periphery. In a second experimentCthe depth-profiled experimenta thin strip of cornea, again cut across the corneal diameter, was orientated so that the X-ray beam passed through the cut edge of the tissue in a direction parallel to the corneal surface. A series of X-ray diffraction patterns, again each 25?m in diameter, was obtained from the epithelial to the endothelial surface of the corneal strip in 50?m steps. This was done for every radial position (from epithelial to endothelial direction) from the centre to the outer periphery of the bovine cornea. Radiation of wavelength em /em ?=?0.83?? was used and diffraction patterns were recorded on 640??480?pixel detector with sub-second exposures. The 67?nm meridional reflection from hydrated rat-tail tendon was used as a calibrant, and the.