Application of second harmonic generation imaging for visualization of the characteristics of corneal stromal collagen in normal and diseased eyes

Second harmonic generation (SHG) is a nonlinear optical phenomenon that occurs when two photons interact with oriented asymmetric molecules. Such molecules in living organisms include collagen, myosin, and tubulin. SHG imaging has thus been applied to the study of these proteins in cells and tissue in vitro or ex vivo. The stroma accounts for about 90% of the thickness of the human cornea and is composed mostly of collagen lamellae formed by oriented collagen fibrils. Approximately 300 such collagen lamellae are layered throughout the thickness of the corneal stroma, with this well-ordered structure being essential for corneal transparency. The corneal stroma also contains a relatively small number of keratocytes. Given the nature of SHG and the structure of the corneal stroma, SHG signals obtained from this tissue are derived specifically from stromal collagen.