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Noninvasive assessment of the hydration gradient across the cornea using confocal Raman spectroscopy
N J Bauer1, J P Wicksted, F H Jongsma
1Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston 77555-0456, USA.
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.