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L. Jay, A. Brocas, K. Singh, I. Brunette, T. Ozaki; High Resolution Non-Invasive Imaging of the Cornea by Epi-Detected Second Harmonic Generation and Third Harmonic Generation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2257. doi: https://doi.org/.
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Advances in femtosecond laser have permitted significant progress in corneal refractive surgery. Additional progress will require non-invasive imaging that offers high spatial resolution across the entire corneal thickness. For clinical applications, imaging in the epi-direction (i.e. from the epithelial side) is also of fundamental importance. In this study, we showed that second harmonic generation (SHG) and third harmonic generation (THG) can be used in combination to perform high resolution imaging of full thickness ex vivo pig corneas.
The laser source used for harmonic generation imaging was a solid-state mode-locked Yb:KGW oscillator (t-Pulse 20; 1030nm, 1000mW, 200fs, 50MHz; Amplitude-Systemes). The nonlinear optical imaging system was an upright microscope built using opto-mechanical parts (AFOptical). Fresh pig eyes were obtained from a slaughterhouse within 6 hours of death and kept in a humid chamber at 4 degrees C until used. In some of these eyes, intrastromal lamellar laser cuts were performed using a chirped-pulse amplification Ti:sapphire laser (KMLabs), in order to document the quality of SHG and THG imaging of these cuts.
SHG and THG signals provided complementary information since second harmonic was generated only in the stroma, while third harmonic was also generated at the level of epithelial and endothelial layers. Both SHG and THG were detected forward and backward accross the entire thickness of the tested corneas, allowing high resolution imaging of the natural structures of the epithelium, stroma and endothelium (e.g. cells, collagen fibres). The laser cuts created by the femtosecond laser were also clearly visualized.
We have demonstrated that the combination of SHG and THG offers unique opportunities for the development of new non invasive microscopy tools for corneal imaging. Moreover, THG offers an alternative to the auto-fluorescence which depends on the presence of NADPH, since third harmonic is generated by any optical inhomogeneities of the cornea. The ability to obtain both SHG and THG signals in epi-detection on fresh eyes gives promising hopes for application of these techniques to clinical studies.
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