A technique called two-photon corneal collagen crosslinking (2P-CXL) has been demonstrated to strengthen the cornea, with results confirmed by confocal Brillouin microscopy. The method could enable selective biological tissue stiffening in situ for applications in ophthalmology, laser surgery and tissue engineering. When treating keratoconus, or weakening or the cornea, an alternative to corneal transplants is one-photon corneal collagen crosslinking (1P-CXL), which is currently used in Europe, Canada and Japan, and is undergoing clinical trials in the U.S. Reconstructed depth profile of riboflavin-soaked corneas before and after one-photon CXL (corneal collagen crosslinking). Green: riboflavin fluorescence, Blue: backscattered collagen SHG (second-harmonic generation). Courtesy of Sheldon J.J. Kwok et al./Optica, a publication of The Optical Society (OSA). “Right now, UV light is used to perform crosslinking across the entire cornea,” said researcher Seok-Hyun Yun of the Massachusetts General Hospital Wellman Center for Photomedicine. “However, this comes with a risk of damaging the innermost layer of the cornea, a complication that changes the corneal function and can cause it to become very hazy. There is also no way to predict whether the procedure will actually improve vision in a given patient.” For this reason, researchers from Wellman sought to qualify two-photon absorption for the precise control of the area of corneal crosslinking. Two-photon absorption uses a near-infrared femtosecond laser to achieve very high spatial resolution confined to a small volume. The approach has been used to harden liquid resins to create microscopic optical components and other 3D structures. Until this research, the technique had not been applied to stiffen tissue, which is initially solid, in a specific area, the researcher said. The Wellman team used a home-built two-photon microscopy setup to deliver femtosecond Ti:sapphire laser light delivered through an objective lens. They placed a cornea sample under the objective lens, applied a light-sensitive dye to the tissue, and then turned on the laser light and focused it to a specific layer. After some trial and error, they found that exposing the tissue to 200-mW laser light for 10 min induced collagen crosslinking without damaging tissue. By scanning the laser through an area of tissue, the researchers said they could induce crosslinking in a specific 3D area. “If you can crosslink a specific part of the cornea, it might be possible to optimize the visual outcome,” Yun said. “With two-photon absorption we can selectively choose and limit the depth of crosslinking to avoid cell damage both on the top and bottom layers of the cornea.” One of the experiment challenges was verifying that crosslinking had taken place, as 2P-CXL is not easily visualized with standard imaging methods. The researchers used Brillouin microscopy to validate that crosslinking was induced in tissue and also showed that the amount of crosslinking produced with two-photon adsorption was almost the same as what was induced with the single-photon approach currently used. Because the two-photon approach induces crosslinking point by point, it is a lengthy process, making it most useful for crosslinking a small region or a thin slice of tissue, the researchers said. Although more studies are needed to understand how various patterns of crosslinking affect corneal shape and vision, the researchers hope that the new technique could allow crosslinking in a 3D pattern specific to a patient’s corneal bulging to maximize the benefits of the treatment. The technique might also be useful for tissue engineering applications to selectively modulate the stiffness of 3D cell cultures to more closely match that of tissue in the body. The research was published in Optica, a publication of The Optical Society (OSA) (doi: 10.1364/optica.3.000469).