Nonlinear microscopy for quantification of riboflavin diffusion in the cornea

Summary Imaging methods are a central part of diagnostics and therapeutic procedures at the anterior segment of the eye. Due to the good accessibility, even high resolution laser scanning technologies are applicable, delivering cellular resolution images of living cells and tissues of the cornea. Using different nonlinear imaging modalities, even diffusion processes at the cornea can be monitored. Using a scanning laser microscope in combination with a tunable optical parametric oscillator, nonlinear images by two‐photon excitation and second‐harmonic generation can be acquired. Further contrast modalities, such as higher harmonics or four‐wave mixing are investigated. Several tissue layers and cellular structures can be visualized in living tissue (mouse model) and ex vivo tissue (pig eyes). Cellular structures as epithelium, endothelium and keratocytes, as well as the lens capsule and cells were imaged, by using second‐harmonic generation and four‐wave mixing, collagen was imaged within the cornea and the transition from cornea to sclera could be studied. Furthermore, diffusion of molecules through the cornea at different concentrations could be monitored as well, for applications such as corneal crosslinking. Nonlinear laser microscopy is a well‐suited method for non‐invasive, staining free imaging of the anterior segment of the eye.