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The cornea is a transparent tissue with significant refractive and barrier functions. The adult cornea has five layers: an outer epithelium layer, a Bowman’s layer which forms the transition between the corneal epithelium and the underlying stroma, a middle stromal layer of collagen-rich extracellular matrix between stromal keratocytes, a Descemet's membrane which separates the stroma from the underlying endothelial layer of the cornea and an inner layer of endothelial cells. Early studies showed that the that collagen fibrils of chick cornea display orthogonal-like pattern. Moreover, primary and secondary stroma have been identified in developing cornea. The primary stroma of the developing avian cornea is a highly organized extracellular matrix composed largely of striated collagen fibrils synthesized by the epithelium. Previous studies have found at least two different fibrillar collagen types, such as type I and II, are shown during the development. Other type of collagen, such as type IX, also appears to be involved in the collagen assembly process. This matrix is subsequently invaded by periocular mesenchymal cells which secrete a secondary cornea stroma to gradually replace the primary one. This indicates that the development of embryonic cornea involves many important biological events and exhibits highly dynamic characteristics. Since second-harmonic generation (SHG) is a nonlinear second order optical process which occurs in noncentrosymmetric systems with a large hyperpolarizability, it has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. It is highly sensitive to the collagen fibril/fiber structure, and to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. To explore the structural variation of embryonic cornea, we use Fast Fourier Transform second harmonic generation microscopy as a tool in our study.
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