June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Collagen Crosslinking By Infrared Femtosecond Pulses In Ex-vivo Corneas
Author Affiliations & Notes
  • Juan Bueno
    Laboratorio de Optica, Universidad de Murcia, Murcia, Spain
  • Harilaos Ginis
    Laboratorio de Optica, Universidad de Murcia, Murcia, Spain
  • Raquel Palacios
    Laboratorio de Optica, Universidad de Murcia, Murcia, Spain
  • Alexandros Pennos
    Laboratorio de Optica, Universidad de Murcia, Murcia, Spain
  • Pablo Artal
    Laboratorio de Optica, Universidad de Murcia, Murcia, Spain
  • Footnotes
    Commercial Relationships Juan Bueno, None; Harilaos Ginis, Universidad de Murcia (P); Raquel Palacios, None; Alexandros Pennos, None; Pablo Artal, AMO (C), Voptica SL (P), Voptica SL (I), AMO (F), Calhoun Vision (F), Calhoun Vision (C), AcuFocus (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5287. doi:
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    • Get Citation

      Juan Bueno, Harilaos Ginis, Raquel Palacios, Alexandros Pennos, Pablo Artal; Collagen Crosslinking By Infrared Femtosecond Pulses In Ex-vivo Corneas. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5287.

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

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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.

Keywords: 484 cornea: stroma and keratocytes • 551 imaging/image analysis: non-clinical • 480 cornea: basic science  

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