Combined nonlinear and femtosecond confocal laser-scanning microscopy of rabbit corneas after photochemical cross-linking

摘要

Purpose. Photochemical cross-linking of corneal stromal collagen using riboflavin and ultraviolet irradiation is an evolving treatment for keratoconus. The purpose of the present study was to investigate the wound-healing process in rabbit corneas after cross-linking. Methods. Photochemical cross-linking was performed according to a standard protocol on the right eyes of eight male New Zealand White rabbits; the left eyes served as controls. Untreated controls and cross-linked rabbit corneas were imaged 3 days, 6 days, and 6 weeks after treatment using a customized setup for three-dimensional nonlinear microscopy and confocal laser-scanning microscopy of reflected femtosecond light (fs-CLSM). Results. The combination of fs-CLSM in reflective mode and two-photon-excited fluorescence permitted differentiation of the following zones in the lamina propria of treated corneas 3 and 6 days after cross-linking: (1) an anterior zone with postapoptotic keratocyte debris, visible only on fs-CLSM in reflective mode; (2) a posterior zone with activated keratocytes with strong autofluorescence; and (3) surviving or restored keratocytes with moderate autofluorescence beyond the intermediate zone. Repopulation with normal keratocytes was achieved by 6 weeks. Bi-directional, second-harmonic generation (SHG) imaging showed no global differences in the fiber orientation and lamellar structure of stromal collagen at any time point. A relatively strong additional two-photon excited fluorescence occurred in the treated corneas with a diffuse three-dimensional spatial distribution. Conclusions. This combination of imaging modalities has the potential to become a new clinical instrument capable of visualizing corneal changes at the cellular and extracellular level.