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.

Purpose

To determine whether riboflavin-induced collagen crosslinking (CXL) could be precisely achieved in the corneal stroma of ex vivo rabbit eyes using nonlinear optical excitation with a low numerical aperture lens and enlarged focal volume.

Setting

Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, USA.

Design

Experimental study.

Methods

The corneal epithelium was removed and the corneas were soaked in 0.5% riboflavin solution. Using a 0.1 numerical aperture objective, a theoretical excitation volume of 150 μm × 3 μm was generated using 1 W of 760 nm femtosecond laser light and raster scanned with 4.4 μm line separation at varying effective speeds over a 4.50 mm × 2.25 mm area. Corneal sections were examined for collagen autofluorescence.

Results

Collagen autofluorescence was enhanced 2.9 times compared with ultraviolet-A (UVA) CXL. Also, increasing speed was linearly associated with decreasing autofluorescence intensity. The slowest speed of 2.69 mm/s showed a mean of 182.97 μm ± 52.35 (SD) long autofluorescent scan lines axially in the central cornea compared with 147.84 ± 4.35 μm for UVA CXL.

Conclusions

Decreasing dwell time was linearly associated with decreasing autofluorescence intensity, approaching that of UVA CXL at a speed of 8.9 mm/s. Using an effective speed of 8.9 mm/s, nonlinear optical CXL could be achieved over a 3.0 mm diameter area in fewer than 4 minutes. Further development of nonlinear optical CXL might result in safer, faster, and more effective CXL treatments.

Financial disclosure

None of the authors has a financial or proprietary interest in any material or method mentioned.

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 μm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ± 3.4% and 47.7 ± 7.6% of the corneal thickness at one and three months. There was a significant (P < 0.05) reduction in CFC in the CAF region in all eyes averaging 1.007 ± 0.006 and 1.009 ± 0.005 in one and three month samples compared to 1.017 ± 0.04 and 1.016 ± 0.06 for controls. These results indicate that there is a significant reduction in collagen crimping following UVA CXL of approximately 1%. One possible explanation for this loss of crimping could be shortening of the collagen fibers over the CXL region.