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Alexander Krüger, Marine Hovakimyan, Diego F. Ramírez Ojeda, Oliver Stachs, Andreas Wree, Rudolf F. Guthoff, Alexander Heisterkamp; Combined Nonlinear and Femtosecond Confocal Laser-Scanning Microscopy of Rabbit Corneas after Photochemical Cross-Linking. Invest. Ophthalmol. Vis. Sci. 2011;52(7):4247-4255. doi: 10.1167/iovs.10-7112.
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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.
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).
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.
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.
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