The purpose of this study was to develop and test a nonlinear optical device to photoactivate riboflavin to produce spatially controlled collagen crosslinking and mechanical stiffening within the cornea. A nonlinear optical device using a variable numerical aperture objective was built and coupled to a Chameleon femtosecond laser. Ex vivo rabbit eyes were then saturated with riboflavin and scanned with various scanning parameters over a 4 mm area in the central cornea. Effectiveness of NLO CXL was assessed by evaluating corneal collagen auto fluorescence (CAF). To determine mechanical stiffening effects, corneas were removed from the eye and subjected to indentation testing using a 1 mm diameter probe and force transducer. NLO CXL was also compared to standard UVA CXL. The NLO CXL delivery device was able to induce a significant increase in corneal stiffness, comparable to the increase produced by standard UVA CXL.

To investigate the role of collagen structure in corneal biomechanics, measurement of localized corneal elasticity with minimal destruction to the tissue is necessary. We adopted the recently developed acoustic radiation force elastic microscopy (ARFEM) technique to measure localize biomechanical properties of the human cornea. In ARFEM, a low-frequency, high-intensity acoustic force is used to displace a femtosecond laser-generated microbubble, while high-frequency, low-intensity ultrasound is used to monitor the position of the microbubble within the cornea. Two ex vivo human corneas from a single donor were dehydrated to physiologic thickness, embedded in gelatin and then evaluated using the ARFEM technique. In the direction perpendicular to the corneal surface, ARFEM measurements provided elasticity values of E = 1.39 ± 0.28 kPa for the central anterior cornea and E = 0.71 ± 0.21 kPa for the central posterior cornea in pilot studies. The increased value of corneal elasticity in the anterior cornea correlates with the higher density of interweaving lamellae in this region.