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It is well established that the state of corneal hydration plays a crucial role in maintaining optimal vision. Therefore, any knowledge that can be obtained non-invasively about the status of corneal hydration could be of significant clinical value. A novel confocal Raman spectroscopic technique was used to monitor non-invasively drug-induced hydration changes in the rabbit cornea. The spectroscopic technique enables one to monitor the changes in water content of the cornea while the confocal probing reduces interference of signals from adjacent tissues and allows for measurement of corneal hydration at various depths. The corneal hydration is altered by applying a dehydrating agent (Muro 128^®) topically on the cornea. To determine the corneal hydration status, the OH/CH ratio between the Raman intensity of the water OH mode at 3390 cm⁻¹ and the protein CH stretching mode at 2945 cm⁻¹ is calculated. In the middle of the corneal stroma after 10 min, Muro 128^®-treated corneas show an average decrease of about 30% in the OH/CH ratio (1.27 ± 0.13) compared with the untreated corneas (1.76 ± 0.09). In this in vivo model it is possible to monitor the hydration status of the living cornea using the Raman spectroscopic technique. Copyright © 2001 John Wiley & Sons, Ltd.

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 vitronear-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 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 (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. © 1997 Society of Photo-Optical Instrumentation Engineers.

The ability of Raman spectroscopy to remotely characterize ocular tissues using a rapid fibre-optic-based system was explored in this study. An argon ion laser operating at 514.5 nm and a charge-coupled-device-based Raman system were used. Raman spectra from aqueous humour specimens of rabbits and humans, as well as spectra from biological molecules obtained through fresh excised rabbit corneas, were obtained. Prominent peaks were observed which qualitatively matched the known locations of biological molecular peaks such as glucose, lactate and urea. In the rabbit aqueous humour spectra, peak positions were also observed indicative of amino acids and proteins. Raman spectra were also obtained from the rabbit lens showing peak positions indicative of amide I and III modes, sulphydryl and CH stretching modes and amino acids. This study demonstrated that it may be possible to characterize ocular tissue structures and to determine in a qualitative manner the presence of various biological molecules in aqueous humour by means of a Raman-based fibre optic instrument. © 1997 by John Wiley & Sons, Ltd.

Individual concentrations of glucose, lactic acid, and urea down to 0.1 wt % have been investigated in aqueous solutions by the Raman scattering technique. These measurements have been conducted with three different laser excitation wavelengths at 514.5, 632.8, and 751.8 nm in conjunction with a single-grating spectrometer and a liquid nitrogen-cooled charge-coupled device (CCD). Rabbit and human aqueous humor specimens have also been studied by this method. In addition to a water background, the large fluorescence structure in the Raman spectra resulting from these aqueous humor specimens has been subtracted out by being fitted to a first-degree polynomial. Raman peaks similar to the stretching vibrations of methylene and methyl groups associated with glucose and lactate, respectively, were observed in the human specimens within the region from 2800 to 3050 cm−1. Similar structure was observed from the rabbit specimens. In addition, a Raman peak, indicative of the carbon-nitrogen stretching mode of urea and various amino acids, was observed at 1006 cm−1in all aqueous humor specimens. However, some of the Raman peaks in the rabbit specimens may have resulted from the euthanasia process.