In Vivo Confocal Raman Spectroscopy of the Human Cornea

PURPOSETo investigate the feasibility of a confocal Raman spectroscopic technique (see ref. 13) for the noninvasive assessment of corneal hydration in vivo in two legally blind subjects. METHODSA laser beam (632.8 nm; 15 mJ) was maintained on the cornea by using a microscope objective lens (x25 magnification, NA = 0.5, f = 10 mm) both for focusing the incident light as well as collecting the Raman backscattered light, in a 180° backscatter configuration. An optical fiber, acting as the confocal pinhole for elimination of light from out-of-focus places, was coupled to a spectrometer that dispersed the collected light onto a sensitive array detector for rapid spectral data acquisition over a range from 2,890 to 3,590/cm. Raman spectra were recorded from the anterior 100–150 μm of the cornea over a period before and after topical application of a mild dehydrating solution. The ratio between the amplitudes of the signals at 3,400/c (OH-vibrational mode of water) and 2,940/cm (CH-vibrational mode of proteins) was used as a measure for corneal hydration. RESULTSHigh signal-to-noise ratio (SNR = 25) Raman spectra were obtained from the human corneas by using 15 mJ of laser light energy. Qualitative changes in the hydration of the anteriormost part of the corneas could be observed as a result of the dehydrating agent. CONCLUSIONWith adequate improvements in system safety, confocal Raman spectroscopy could potentially be applied clinically as a noninvasive tool for the assessment of corneal hydration in vivo.