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

Invest Ophthalmol Vis Sci. 1998 Apr;39(5):831-5.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biological Dressings
  • Body Water / metabolism*
  • Collagen / metabolism
  • Cornea / metabolism*
  • Female
  • Microscopy, Confocal
  • Rabbits
  • Spectrum Analysis, Raman / methods


  • Collagen