Sodium fluorescein as a retinal pH indicator?

Retinal neovascularization is a symptom associated with various diseases revealing ocular fundus manifestation. Often, these neovascularizations originate from retinal hypoxia. A concomitant phenomenon of hypoxia is acidosis. To recognise this would permit the identification and treatment of hypoxic fundus areas long before first vascular modifications are seen. Thus, the goal of this investigation was to elucidate whether sodium fluorescein could be used as a retinal pH indicator. Sodium fluorescein solution was diluted in PBS (ratio: 1: 150 000). The pH was varied from 6.5 to 8.6 by supplementation of HCl or NaOH, respectively. The fluorescence was excited by a pulsed diode laser (wavelength: 446 urn, pulse width: 100 ps) and detected by time-correlated single photon counting (TCSPC) technique. A least-squares fit of the measured fluorescence decay versus time by an exponential function results in the fluorescence lifetime. Ten measurements were taken at each pH for statistical analysis. The dependence of the fluorescence lifetime on the temperature and the concentration of sodium fluorescein was investigated in the same way. The fluorescence lifetime was found to rise from 3.775 ns to 4.11 ns with increasing pH (6.5 to 8.6). However, the gradient decreases with increasing pH. We found highly significant differences (Student's t-test, P < 0.0005) of the fluorescence lifetimes for pH values with a mean difference of 0.125 at pH < 7.65 whereas the differences were still significant (P <= 0.02) at pH > 7.65 and mean pH differences of 0.2. The fluorescence lifetime was independent of the temperature (22 degrees C to 37 degrees C) and the concentration of sodium fluorescein (dilution 1: 150 000 to 1:2000). The fluorescence lifetime of sodium fluorescein depends on the pH but not on temperature and concentration. Thus, the discrimination of areas with retinal acidosis should be possible by combination of the TCSPC technique with scanning laser ophthalmoscopy. Further investigations have to clarify whether the accuracy of the measurement at the fundus in vivo is sufficient.