Abstract
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.
Highlights
The cornea is the outer part of the eye that protects against external injuries and contributes to 2/3 to the eye refractive power
FSHG images are characterized by striated spatial features, while B-Second Harmonic Generation (SHG) images are nearly homogeneous at the micrometer scale, with speckle-like background interrupted by linear cracks with much smaller signal
Similar features are observed in transverse reconstructions of forward SHG (F-SHG) and backward SHG (B-SHG) images (Figs. 3C-D and Media 2)
Summary
The cornea is the outer part of the eye that protects against external injuries and contributes to 2/3 to the eye refractive power Available techniques, such as confocal reflectance microscopy [1] or optical coherence tomography (OCT) [2,3], enable threedimensional (3D) cell-scale imaging of cornea. These techniques lack specificity or contrast when looking at the collagen organization of the corneal stroma. Each lamella forms 10-100 μm wide domains that are comprised of 30 nm diameter collagen fibrils organized into a hexagonal lattice This highly organized structure is responsible for both the transparency and the mechanical strength of the cornea. It is of great interest to develop in vivo imaging techniques that provide structural and quantitative information about the corneal stroma
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