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Purpose: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFM)., Methods: A normal eye removed from a 66-year-old male was used in the study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a non-contact mode AFM in air., Results: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 +/- 1.0 (mean +/- standard deviation) nm and the scleral fibrils of 82.5 +/- 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found in both types of fibrils; the D-periodicity and the groove depth were 65.7 +/- 0.8 nm and 1.46 +/- 0.50 nm in the corneal fibrils, and 67.3 +/- 1.1 nm and 6.16 +/- 1.23 nm in the scleral fibrils., Conclusions: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution.

We studied scleral specimens from experimentally induced enlarged eyeball with axial elongation by the transmission electron microscopy following cationic dye (cuplonic blue) staining. The animal model was prepared by the injection of alpha-chymotrypsin into the posterior chamber of young albino rabbits. Cuprolinic blue staining was applied to scleral specimens obtained from equatorial lesion and portions of the scleral tissues were subjected to enzyme digestion by chondroitinase ABC, AC and B before cuprolinic blue staining. In control eyes, dermatan and chondroitin type sulfated proteoglycan filaments were identified. Large, electron dense, and abnormal by shaped proteoglycan filaments were seen in the transmission electron microscopy. Such abnormal proteoglycan filaments were susceptible to enzyme chondroitinase ABC and AC digestion but resistant to chondroitinase B, suggesting that they are chondroitin sulfate dominant proteoglycans. Our morphological data corroborated a previous biochemical report of abnormally induced proteoglycan molecules in sclera with enlarged, axially elongated eyes.

We examined a scleral specimen obtained from a nanophthalmos eye with uveal effusion at the surgery, using cuprolinic blue (CB) dye binding and electron microscopy. The nanophthalmic sclera was separated into two areas electron microscopically. One was an area composed of tightly packed collagen fibers, the other with irregularly arranged collagen fibers. In irregular areas, several abnormal findings, such as twisting or fraying, were detected in collagen fibers, which were surrounded by fine fibrous materials. Furthermore, CB-positive materials were fewer in number and less in size and electron density around the collagen fibers and in fine fibrous materials than in the normal eye. Since the nanophthalmic sclera in this study showed some abnormalities both in collagen fibers and in proteoglycans, we suggest that the function and/or interaction of both of these were abnormal, which might be related to the onset of uveal effusion.