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Revealing Corneal Stroma Organization With Two-Photon Microscopy


Purpose: : To further investigate the organization of stroma in healthy corneas of different species from tomographic images of ex-vivo samples obtained with a two-photon microscope.

Methods: : A custom two-photon (non-linear) microscope has been used to study the structure of corneas of different species: porcine, bovine, rabbit, rat, chicken and humans. The instrument uses a Ti:Sapphire femtosecond laser for illumination, a scanning unit and a photon-counting detection device. Second harmonic generation (SHG) signals produced by collagen in the corneal stroma were registered in a reflection configuration. A motorized stage allowed optical sectioning across the entire corneal thickness. Series of XY images were used for tomographic (3D) reconstruction of the corneas, revealing the detailed stromal architecture with 0.5 microns lateral and 2 microns axial resolutions. Samples were neither fixed not stained and they were fully in-depth scanned.

Results: : SHG images revealed the microscopic organization of the lamellae of collagen fibers. Despite absorption, for all corneal depths, images were good enough to be analyzed. The anterior stroma was similar in all samples, showing interwoven short bands of collagen randomly distributed. The lamellae at the central and posterior stroma were densely packed and often presented longer bundles lying predominantly parallel to the corneal surface with characteristic spatial distributions for each species. In particular, collagen bundles in bovine and porcine corneas were interweaved and presented undulations. In human corneas, lamellaes were longer and had similar orientation than their neighbours. In the chick cornea, the stromal arrangement had an orientation changing regularly with depth.

Conclusions: : Two-photon microscopy provided non-invasively information on the corneal morphology through backscattered SHG images. Changes of the stromal collagen organization within the corneal depth were characterized for different animal models and human corneas.

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