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Giuliano Scarcelli, Sabine Kling, Elena Quijano, Roberto Pineda, Susana Marcos, Seok Yun; Brillouin microscopy of collagen crosslinking: non-contact depth-dependent analysis of corneal elastic modulus. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4075.
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Corneal collagen crosslinking (CXL) is a promising method to treat keratoconus and corneal ectasia. The increase of corneal rigidity produced by CXL is a determinant of treatment outcome. However, it is currently difficult to monitor and objectively evaluate the mechanical outcome due to the lack of suitable methods for measuring the mechanical properties of the cornea in vivo. The objective of this study is to test and validate a recently developed Brillouin microscopy technique for quantifying the corneal mechanical properties before and after CXL.
CXL was performed on N=21 fresh porcine eyes using various pre-soaking times, light doses, and with (riboflavin-mediated) or without (benzalkonium chloride -mediated) epithelial debridement. A confocal Brillouin microscope was used to measure the Brillouin elastic modulus maps of the corneas without contact before and after CXL. Brillouin corneal stiffness was calculated from the Brillouin cross-sectional maps. The average elastic moduli of anterior, middle, and posterior stromal regions were analyzed for different CXL protocols. Corneal Stiffening Index (CSI) was introduced as a metric to compare the efficacy of a given CXL protocol with respect to the standard Dresden protocol (30-min pre-soak of riboflavin, ultraviolet illumination for 30 min at 3 mW/cm2).
Brillouin corneal stiffness increased significantly (P<0.001) by both epi-off and epi-on CXL. The increase of Brillouin modulus was depth-dependent, decreasing from the anterior to posterior regions, indicating that the stiffening of the anterior corneal stroma contributes the most to mechanical changes. The increase of anterior Brillouin modulus was linearly proportional to the light dose. Compared to the standard epi-off procedure, a typical epi-on procedure resulted in a third of stiffness increase (i.e. CSI = 33).
Brillouin microscopy allowed imaging and quantifying CXL-induced changes of elastic modulus, without contact, in a depth-resolved manner, and at a high spatial resolution. This technique may be useful in evaluating the mechanical outcomes of CXL procedures as well as comparing different protocols and CXL agents. This work paves the way towards an objective real-time monitoring of CXL in clinical and in experimental settings.
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