Confocal Raman spectroscopy system for noncontact scanning of ocular tissues: an in vitro study

A long-working-distance fiber-optic-based confocal Raman spectroscopy (CRS) system, operating in the backscatter mode, was developed for rapid noninvasive characterization of ocular tissue. In vitro near-real-time axial scanning through ocular tissue was achieved using a CCD camera and a high-numerical-aperture long-working-distance microscope objective in a telecentric configuration. The system provides high spatial resolution (20 to 150 mu m) Of transparent ocular tissues up to 13 mm deep into the eye in a noncontact fashion while utilizing low argon-laser power and rapid scanning times (25 mJ), yielding a SNR range from 30 to 75. To test the performance of the system for characterizing ocular tissue, Raman spectra from rabbit eyes were obtained in vitro. Axial scans of the cornea, the aqueous humor, and the lens provided discrete and specific Raman spectra from each tissue, in both the lower and the higher wave-number region. Characteristic Raman signals common to all tissues are the OH vibrations (1650 and 3100 to 3700 cm(-1)) and the vibrations corresponding to amino acids (phenylalanine at 1003 cm(-1), tryptophan at 760 and 881 cm(-1), and tyrosine at 646 cm(-1)). The ocular lens can be identified by three distinct peaks (aromatic and aliphatic CH stretching and OH bending modes), of which the aromatic CH stretching mode (approximate to 3057 cm(-1)) is lens-specific. The cornea can be identified by the presence of two distinct peaks (aliphatic CH stretching and OH bending) and the absence of the aromatic CH stretching mode, The aqueous humor can be identified by the presence of the OH bending mode and the lack of the both CH stretching modes. A long-working-distance confocal Raman spectroscopy system may offer a novel technique for the noncontact spatially resolved biochemical characterization of various tissue layers of the anterior segment of the eye. (C) 1997 Society of Photo-Optical Instrumentation Engineers.