Abstract
Diseases that affect the cornea can lead to severe vision loss and have tremendous social impact. These diseases are associated to deviations from normal structural order and orientation of collagen fibril bundles. Unfortunately, resolving non-invasively the corneal collagen structure is not possible to date. In this work, polarization sensitive second harmonic generation (pSHG) microscopy is used to obtain information with molecular specificity on microstructure of human corneas. This information is used to develop a set of label-free imaging biomarkers that were generated by means of a novel methodology based on mathematical tensorial calculus. The method is proven to be highly sensitive and robust. The use of these biomarkers permits accurate characterization of the anisotropic, depth-dependent, structural organization of corneal collagen fibril bundles without any a priori information. The method can be valuable to improve understanding of microstructural pathophysiological changes of the human cornea close to in vivo conditions.
Highlights
The cornea is the main refractive element of the human eye
We demonstrated the use of polarization sensitive second harmonic generation (pSHG) microscopy for providing valuable descriptors of the human corneal structure based on the acquired images; these images were processed using methods based on a tensorial mathematical approach taken from liquid crystal theory
The novel pSHG imaging biomarkers, f, S, and T were calculated for each plane within each corneal sample
Summary
Its main structural component is type I collagen. The shape and transparency of the cornea are maintained by the highly organized assembly of stromal collagen. These are embedded in an optically homogeneous ground substance, the stromal matrix, consisting of glycosaminoglycans and proteoglycans [2]; in addition, they show a highly heterogeneous distribution over the stromal depth. Collagen fibril bundles in the most anterior stroma show pseudo-random spatial distribution; across the posterior stroma, they are arranged in lamellae that run almost parallel to the corneal surface, forming a grid-like lamellar structure with more ordered organization if compared to the anterior stroma. Clinical benefit of resolving the microstructure of the corneal stroma would extend to several applications, such as screening of keratoconus (the first cause of corneal transplantation in young people worldwide), precise understanding of corneal scarring (first cause of corneal blindness worldwide) and monitoring of the effect of corneal surgery (excimer laser surgery, femtosecond laser assisted lamellar corneal transplantation) [3,4]
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