Collagen cross-linking (CXL) with ultraviolet (UV) A light and riboflavin technique has been reported to increase corneal stiffness to slow down keratoconus progression. With UV CXL treatment, irradiation is not localized and the penetration depth depends on the attenuation of the UV light propagating through the cornea due to absorption within the riboflavin. This fact may lead to endothelial cell loss, especially in thin corneas. The purpose of this work was to determine if femtosecond (fs) infrared pulses could induce localized CXL effects through a 2-photon process on ex-vivo porcine corneas.

A custom-made multiphoton microscope was used to irradiate a corneal volume and subsequently record multiphoton images of the stroma. After de-epithelization, the eye was immersed into a solution containing 0.125% riboflavin and 20% dextran during 45 minutes. A set of control eyes were only immersed into 20% dextran solution in order to maintain their normal hydration. An area 90×90×50 μm³ within the cornea (depth location 125 μm, scanning step 2 μm) was irradiated with the 760-nm fs-laser for 60 minutes in both control and fs-CXL treated eyes.

During irradiation, multiphoton tomographic (XZ or YZ) images of the corneal stroma were adquired every 10 minutes for both control and fs-CXL-treated corneas. For the former the nonlinear signal presented a maximum close to the Bowman’s layer and a uniform decrease with depth typical of a non-irradiated cornea. On the opposite, fs-CXL corneas exhibited a marked increase in nonlinear signal at the treated volume with no effect on nearby corneal layers, presumably indicative of localized changes in collagen distribution. This was revealed by means of the regular XY multiphoton images at different depth positions throughout the entire stroma.

This study demonstrates the feasibility of using infrared fs-laser pulses to perform well-localized 3-dimensional CXL treatments within the cornea. The technique has been proven to be precise with surrounded regions hardly affected and no thermal effects. The combination of both surgical and monitoring functions into a unique fs-laser system has the potential to be a tool in clinical environments and could be the basis to establish a new technique capable of monitoring CXL procedures in real-time.