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.
SHG microscopy was successfully used to characterize the orientation of sutural lamellae within corneal samples by means of an epi-detection scheme. In particular, the attention was focused on the organization and orientation of corneal collagen lamellae in the first 30 μm of the stromal layer below Bowman’s membrane. In fact, it is thought that the irregular orientation of these specific lamellae, an in particular the existence of so-called sutural lamellae, affects the mechanical properties of the cornea and is responsible of the overall corneal stiffness. 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. These samples represent a good benchmark for testing the methodology, considering that keratoconus is an ophthalmic disease in which the cornea loses its stiffness and acquires an abnormal conical shape because of a different organization of sutural lamellae. The samples were first imaged and characterized on the basis of 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.
Keratoconus is an eye disorder that features a reduced stiffness of the cornea and its consequent pathological
deformation. Cross-Linking (CXL) treatment has proven useful in hindering the progression of keratoconus, offering a
minimally-invasive alternative to corneal surgical transplantation. In this study, the biomechanical characteristics of a
human keratoconic cornea were clinically examined in vivo soon before keratoplasty, and the morphological alterations
of the collagen scaffold in the same cornea were examined ex vivo by means of Second-Harmonic Generation (SHG)
microscopy. A healthy cornea and a CXL-treated keratoconus were compared. In particular, the lamellar organization in
the three corneal samples was characterized in different stromal layers by detecting both forward- and backwardscattered
SHG signal and then considering the forward/backward (F/B) ratio as parameter. The F/B ratio was used to
characterize the morphological organization of collagen lamellae within different stromal layers, finding an increased
disorder at the level of Bowman's membrane, opposed to a more regular organization within deeper stromal layers in all
the examined samples. The organization of collagen lamellae in CXL-treated keratoconic samples was similar to that one
found in healthy corneas, demonstrating that the CXL is able to rearrange the collagen scaffold and partially recover the
properties of a healthy condition. The obtained results are in agreement with previous results obtained in studies aimed at
monitoring the organization of fibrillar collagen using F/B SHG ratio. In conclusion, the proposed method might be
useful for both diagnosing keratoconus as well as for monitoring the effects of the CXL treatment.
Keratoconus is an ophthalmic disease in which the cornea acquires an abnormal conical shape that prevents the correct
focusing on the retina, causing visual impairment. The late diagnosis of keratoconus is among the principal causes of
corneal transplantation surgery. In this study, we characterize the morphology of keratoconic corneas by means of the
correlation of SHG images, finding that keratoconus can be diagnosed by analyzing the inclination of lamellae below
Bowman’s membrane. In addition, imaging performed with both sagittal and “en face” geometry demonstrated that this
morphological features can be highlighted both ex vivo and in vivo.
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