Comparison of morphological changes of corneal collagen fibers treated with collagen crosslinking agents using second harmonic generation images.

Corneal cross-linking (CXL) is a common surgical procedure used to modify corneal biomechanics and stabilize keratoconus progression which is still under discussion. Its side effects, which are mostly related to anatomical unpredictability and stromal exposure, are the reason for the search for new CXL agents. In this work we have quantitatively evaluated the porcine corneal stroma architecture treated with collagen crosslinking agents such as riboflavin solutions and açai extract, using second harmonic generation microscopy. Aimed at evaluating the morphological changes in the corneal stroma after collagen crosslinking under a CXL chemical agent, a tubeness filter based Hessian matrix to obtain a 3D fiber characterization of the SHG images was applied. The results showed a curling effect and shortening of the collagen fibers treated with açai as compared to the control. They also showed a higher degree of clustering of the collagen fibers with larger empty spaces when compared to the other two groups. We believe that studies such as these presented in this paper are a good direct nondestructive and free labeling evaluation technique that allows the observation of morphologic features of corneas treated with new CXL agents.