Time-resolved fluorescence properties of fluorescein and fluorescein glucuronide

The use of fluorescein as a tracer in the study of the blood-ocular barriers is complicated by the metabolic production of fluorescein monoglucuronide, the excitation and fluorescence spectra of which overlap with those of fluorescein. Time-resolved fluorescence measurements provide a means of detecting the two substances in a mixture. Fluorescein and fluorescein glucuronide have different fluorescence lifetimes, 4.0 nsec and 2.3 nsec, at 37 degrees C and pH 7.35. Hence the two substances can be distinguished from the biexponential fluorescence decay in the mixture. The lifetimes are identical in buffered water, in hyaluronic acid and in human vitreous in vitro. The method is suggested for estimation of fluorescein and fluorescein glucuronide in the human vitreous in vivo. The time-resolved and steady state fluorescence anisotropies, the fluorescence lifetimes and the quantum yields strongly suggest that binding of fluorescein or fluorescein glucuronide to hyaluronic acid or other macromolecules in the human vitreous is weak, if present at all, and that static quenching of fluorescence does not occur in the vitreous.