Corneal and scleral collagens—a microscopist’s perspective.

This paper reviews our existing understanding of the distribution and organisation of collagen types within the corneal and scleral stroma from a microscopical perspective. The contribution of various types of light microscopy, electron microscopy and atomic force microscopy to this field are separately discussed. Light microscopy was used in the earliest studies of the cornea and lead to the first description of the lamellar structure of the stroma. More recently polarised light microscopy has been used to obtain specific information on fibril orientation within individual lamellae. Light microscope immunolabelling techniques have been utilised to determine the distribution of several collagen types within the cornea and sclera, while recent developments in confocal microscopy have allowed detailed observations to be made on live cornea. Scanning electron microscopy has proved useful in determining the 3D organisation of lamellae within both corneal and scleral stroma. The transmission electron microscope was responsible for first revealing the regular diameter and high degree of order of the collagen fibrils present in the corneal stroma and contrasting this with the irregular diameter of fibrils present in sclera. This finding lead directly to the formulation of a theory of corneal transparency based on the uniformity of fibril diameter and packing. The use of specialised stains such as cuprolinic blue allowed direct observation of the glycosaminoglycan chains on proteoglycan molecules in cornea and sclera. These images allowed the binding sites of the proteoglycans along the collagen fibrils to be identified and provided convincing evidence for the importance of the proteoglycan molecules in collagen fibril organisation. Immunogold labelling has been used to map the distribution of several collagen types within the corneal and scleral stroma at the ultrastructural level and provided critical evidence for the role of type V collagen in the regulation of fibril diameter within the cornea. Specialised freezing–etching techniques have revealed the surface features of the collagen fibrils in corneal stroma, indicating clearly the presence of crossbridge structures between fibrils. The technique of rotary shadowing has been used to determine the conformation of several collagen types. In more recent years atomic force microscopy has been applied to the study of the corneal stroma. It has largely confirmed the observations made by the transmission electron microscope and provided independent evidence of crossbridge structures between the collagen fibres in cornea and sclera. The full potential of this technique has yet to be realised