Water exchange in human crystalline lens studied by combined dispersion confocal microspectroscopy

We have employed the technique of the confocal Raman microspectroscopy to monitor water exchange in human eye lens in vitro. Heavy water (D2O) was used as a marker of the exchange. Raman spectra in the high frequency range (2200-3500 cm-1) were successively recorded at several locations inside the lens in order to register dynamics of the replacement of H2O by D2O. The intensities of the Raman peaks at 2450 cm-1 (OD stretch) and 3390 cm-1 (OH stretch) were used as indicators of H2O/D2O exchange, whereas the Raman peak at 2935 cm-1 (CH stretch of protein) served as an internal standard. The ratios of the Raman intensities I2450/I2935 and I3390/I2935 determined the relative concentrations of D2O and H2O, respectively. For the quantitative description of the exchange, we proposed a diffusion model assuming a constant diffusion coefficient over the volume of the lens. We report the results of experiments performed on four fresh and one fixed lenses. The diffusion coefficient of D2O in the human eye lens was estimated using the least-squares fit of the experimental data.