In situ determination and matching of the refractive index of the human cornea to improve polarization-resolved SHG imaging in depth.

The human cornea is a highly organized tissue, which comprises hundreds of 1-3 µm thick stacked collagen lamellae. However, this microstructure is poorly characterized and requires further investigation. Polarization-resolved second harmonic generation (pSHG) microscopy is a powerful technique for this purpose because of its specificity for collagen and its sensitivity to its orientation. However, pSHG is prone to spatial resolution degradation with depth unless the immersion refractive index is matched to that of the sample, which is critical for corneas that are approximately 600 µm thick. In the absence of experimental data on the refractive index along the entire cornea, we propose a measurement method that applies to the entire cornea directly under the microscope objective. We then use an iodixanol solution to match the refractive index of the immersion medium to that of the cornea. Finally, we carefully characterize the pSHG orientation data obtained under these optimal conditions, and we show that they provide a better resolution along the entire thickness of the cornea and a better determination of the lamellae orientation.