Second-harmonic generation microscopy of photocurable polymer intrastromal implants in ex-vivo corneas.

A custom adaptive-optics (AO) multiphoton microscope was used to visualize the corneal stroma after the insertion of a photocurable polymer material. A lamellar pocket was created and a certain amount of polymer in liquid form was injected. This turned into a rigid film after UV irradiation. Intact eyes were used as control. Tomographic and regular second harmonic generation (SHG) microscopy images were recorded from both control and corneas with polymer implants. In control corneas, the SHG signal decreased uniformly with depth. However, treated corneas exhibited an abrupt loss of SHG signal at the implant location. The use of AO increased the SHG levels and improved the visualization of the stroma, not only at deeper corneal layers but also beneath the implant. Moreover, the absence of SHG signal from the implant allowed its geometrical characterization (thickness and location). This technique offers a potential tool for non-invasive analysis of morphological changes in the cornea after surgery or treatment, and might be useful in future clinical environments.