Three-dimensional mapping of corneal lamellar orientation by means of backward-scattered SHG microscopy

SHG microscopy was successfully used to characterize the orientation of sutural lamellae within corneal samples by means of an epi-detection scheme. Sutural lamellae are located in the first 30 μm of the stromal layer below Bowman's membrane; they affect the mechanical properties of the cornea and are responsible of the overall corneal stiffness. This stromal layer is particularly interesting for diagnosing and following-up keratoconus, an eye disorder that features a reduced stiffness of the cornea and its consequent pathological deformation. The methodology was tested by acquiring image stacks of the central portion of the cornea in ex vivo samples of healthy corneas, keratoconic corneas and keratoconic corneas treated with cross-linking (CXL), a treatment aimed at hindering the progression of keratoconus. The samples were imaged and characterized based on forward/backward SHG ratio, finding a different ratio in keratoconus with respect to both the healthy cornea and cross-linked keratoconus. The inclination of corneal sutural lamellae was then characterized by means of a three-dimensional correlation analysis on SHG images, acquired using a backward detection geometry. Such method provided good discrimination capabilities, demonstrating that this approach can be used not only for diagnosing keratoconus in a very early stage, but also for performing treatment follow-up on cross-linked corneas.