June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Rapid, Single Pass Non-linear Optical Collagen Crosslinking (NLO CXL) of Ex Vivo Rabbit Eyes
Author Affiliations & Notes
  • Samantha Bradford
    Gavin Herbert Eye Institute, Irvine, CA
  • Donald Brown
    Gavin Herbert Eye Institute, Irvine, CA
  • Tibor Juhasz
    University of California, Irvine, Irvine, CA
  • James V Jester
    Gavin Herbert Eye Institute, Irvine, CA
  • Footnotes
    Commercial Relationships Samantha Bradford, None; Donald Brown, None; Tibor Juhasz, None; James Jester, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3007. doi:
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      Samantha Bradford, Donald Brown, Tibor Juhasz, James V Jester; Rapid, Single Pass Non-linear Optical Collagen Crosslinking (NLO CXL) of Ex Vivo Rabbit Eyes. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3007.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: In previous studies we have shown that crosslinking of collagen hydrogels can be achieved using NLO CXL with highly focused, 760 nm femtosecond (FS) laser light. While NLO CXL produces localized and depth controlled crosslinking, highly focused laser light requires multiple passes to achieve a large CXL volume. The purpose of this study was to determine whether NLO CXL could be achieved in intact ex vivo rabbit eyes using a low NA lens for rapid, single pass, enlarged focal volume CXL.<br />

Methods: Rabbit eyes (18) were shipped to the laboratory fresh, the epithelium removed and the corneas soaked in 0.5% Riboflavin/PBS with dextran (20%). Using a Zeiss LSM 510 microscope with a 4x, 0.1 NA objective a theoretical two photon excitation volume of 150 µm axial and 3 µm lateral was generated using 1W of 760 nm FS laser light. Corneas were then raster scanned with 4 µm line separation at varying pixel dwell times over a 4.5 by 2.25 mm area. Treated and control corneas were then sectioned perpendicular to the line of CXL and examined for collagen auto fluorescence (CAF) in the region of 400-450 nm using the Zeiss Meta Detector. CAF was then quantified by subtracting the average pixel intensities of the treated and untreated regions in a section. For comparison, CAF was also measured in normal and standard Dresden UVA CXL rabbit eyes.

Results: Single pass, NLO CXL at the greatest dwell time of 1.63 ms showed 3 μm wide by 180 μm long CAF scan lines in the central cornea. These volumes approximated the theoretical TP focal volumes calculated for a 0.1 NA objective. Intensity of CAF signal from NLO CXL was also 2.9 times greater than that achieved by standard UVA CXL and of equivalent thickness averaging 182.97 ± 52.35 µm for NLO CXL and 147.84 ± 4.35 µm for UVA CXL. Decreasing laser dwell time was associated with a linear decrease in CAF intensity that approached the UVA CXL CAF intensity at 600 µs dwell time.

Conclusions: Using a low NA lens, single pass NLO CXL can induce greatly enhanced CAF of equal depth in the cornea compared to standard UVA CXL. This approach also provides for rapid NLO CXL at a rate of 31 s/mm2 and thickness of 182.97 ± 52.35 µm, allowing a circular 3 mm diameter area to be scanned in <4 min. Further development of NLO CXL may provide for a more safe and effective corneal crosslinking that would be advantageous for LASIK patients.


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