Project Summary/Abstract Collagen crosslinking (CXL) using single photon, 380 nm, UVA light to photoactivate riboflavin (Rf) is known to induce a two-fold increase in collagen stiffness and 1-3 diopters of corneal flattening. While these effects have been shown to have significant therapeutic benefits for both treating Keratoconus and correcting minor refractive errors, there are several major drawbacks to this procedure, including post-operative pain and and delayed visual recovery with increased risk of bacterial infection and corneal scaring. In our recently completed, NEI funded research project we developed a two-photon, nonlinear optical (NLO) CXL approach using infrared femtosecond laser (FS) light that limits Rf photoactivation to the two-photon focal volume of an objective lens. This device uses an amplified FS laser to generate 760 nm, 0.3 μJ pulses at less than 46.1 mW total power (under the ANSI thermal limit for laser eye exposure), and can crosslink a 4 mm diameter area in less than 4 min, significantly enhance stromal stiffness 1.6 fold, and induce 1 to 2 diopters of corneal flattening in live rabbits. As part of this project, we have also developed a novel, FS laser based, corneal epithelial micromachining approach to create Microchannels (MicroCh) through the epithelium to greatly enhance transepithelial (TE) Rf diffusion. Goal of this project is to test the hypothesis that post-operative pain and delayed visual recovery is due to the excessive cellular damage caused by UVA CXL and that TE-NLO CXL shows significantly less cell damage with faster visual recovery. This hypothesis will be tested through the following specific aims: Aim 1. Optimize MicroCh TE Rf stromal penetration by determining the effects of pulse energy, density and depth on Rf stromal concentration and epithelial/stromal integrity as compared to other excipient TE approaches using ex vivo rabbit eyes. Aim 2. Determine the effects of TE Rf penetration on corneal epithelial and stromal integrity comparing MicroCh to optimal excipient Rf approaches identified in SA1 using an ex vivo rabbit eye model to measure epithelial/stromal cell death and induced inflammatory gene expression at 3 hours and 24 hours with and without UVA or NLO CXL. Aim 3. Determine the effects MicroCh Rf delivery and UVA versus NLO CXL in live rabbits by assessing the effects on epithelial integrity, corneal sensitivity, stromal inflammation, wound healing and visual recovery.