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Purpose:

To investigate the organization of stromal collagen in healthy ex vivo corneas of different species from second harmonic generation (SHG) microscopy images.

Methods:

A custom backscattered nonlinear microscope has been used to study the corneal structure of different species: porcine, bovine, rabbit, rat, chicken, and humans. The instrument uses a femtosecond laser for illumination, a scanning unit, and a photon-counting detection device. It also includes a wavefront aberration control module. SHG signals produced by collagen within the cornea were acquired. A motorized stage allowed optical sectioning across the entire corneal thickness. Samples were neither fixed nor stained, and they were fully scanned.

Results:

SHG images revealed the microscopic organization of the lamellae of collagen fibers. Despite absorption, for all corneal depths, images could be analyzed. The anterior stroma was …