In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy

The transparency and mechanical strength of the cornea are related to the highly organized three-dimensional distribution of collagen fibrils. It is of great interest to develop specific and contrasted in vivo imaging tools to probe these collagenous structures, which is not available yet. Second Harmonic Generation (SHG) microscopy is a unique tool to reveal fibrillar collagen within unstained tissues, but backward SHG images of cornea fail to reveal any spatial features due to the nanometric diameter of stromal collagen fibrils. To overcome this limitation, we performed polarization-resolved SHG imaging, which is highly sensitive to the sub-micrometer distribution of anisotropic structures. Using advanced data processing, we successfully retrieved the orientation of the collagenous fibrils at each depth of human corneas, even in backward SHG homogenous images. Quantitative information was also obtained about the submicrometer heterogeneities of the fibrillar collagen distribution by measuring the SHG anisotropy. All these results were consistent with numerical simulation of the polarization-resolved SHG response of cornea. Finally, we performed in vivo SHG imaging of rat corneas and achieved structural imaging of corneal stroma without any labeling. Epi-detected polarization-resolved SHG imaging should extend to other organs and become a new diagnosis tool for collagen remodeling.