Noninvasive assessment of the hydration gradient across the cornea using confocal Raman spectroscopy.

PURPOSE The feasibility of Raman spectroscopy for the noninvasive assessment of axial corneal hydration was investigated. METHODS A scanning confocal Raman spectroscopy system, with an axial resolution of 50 microns, was used to assess noninvasively the water (OH-bond) to protein (CH-bond) ratio as a measure of the hydration in collagen-based phantom media and rabbit corneas. RESULTS Raman spectra with high signal-to-noise ratios were obtained under in vitro and in vivo conditions within a range of corneal hydration (H = 0.0-8.3 mg water/mg dry wt). The Raman intensity ratio OH/CH showed a strong correlation with the hydration of the phantom medium (R2 > 0.99) and the rabbit corneas (R2 > 0.95). A degree of reproducibility was seen in measurements performed at a specific depth within the cornea (SD = 1.2%-2.7%). Quantitatively, the spatially resolved corneal water content, as assessed with our method, showed an increasing gradient from the anterior to the posterior region, with a difference of approximately 0.9. Significant qualitative differences in the axial hydration gradient were observed between the in vitro and in vivo situation, caused by the presence of an intact tear-film in vivo. Characterization of the axial corneal hydration using Raman spectroscopy provided a reliable estimation of total corneal hydration compared with conventional measurements using pachymetry and lyophilization. CONCLUSIONS The proposed noninvasive confocal Raman spectroscopic technique has the potential to assess the axial corneal water gradient with a degree of sensitivity and reproducibility.