Optical Biopsy of Ocular Tissue with Two-Photon Excitation Laser Scanning Microscopy

Abstract

Two-photon excitation laser scanning microscopy is used to produce three-dimensional maps of cellular metabolism based on the fluorescence of the naturally occurring reduced pyridine nucleotides NAD(P)H. The fluorescence from NAD(P)H was imaged with submicron lateral resolution through the 400 micron thickness of the cornea. Metabolic imaging with two-photon excitation scanning laser microscopy with near-infrared excitation has several advantages over conventional ultraviolet light. The near infrared light can penetrate deeper into the ocular tissue, there is reduced photodamage, and the chromatic aberration that occurred with ultraviolet excitation light is eliminated. In order to confirm that the fluorescence intensity is predominately from the NAD(P)H, the tissues were incubated with cyanide. A subsequent time dependent doubling of the intensity of fluorescence resulted. All cell types in the cornea of an ex vivo eye were imaged. The fluorescence from keratocytes in the corneal stroma was only observed after cyanide treatment. NAD(P)H fluorescence from lens epithelial cells was observed as well as from the lens fibers. These studies demonstrate the validity of using two-photon excitation laser scanning microscopy to perform a noninvasive optical biopsy of ocular tissue.