Fluorescence properties of dibenzofluorescein in aqueous solution.

The deprotonation of dibenzofluorescein (DBFL), a long wavelength fluorescence probe, results in the simultaneous occurrence of neutral form, monoanion and dianion under physiological conditions. The fluorescence properties of the former two cannot be measured directly because they are always coexistent with some other species. By measuring the fluorescence under various pHs we computed the fluorescence parameters for each species involved in the prototropic equilibria of DBFL, including each species' emission spectrum, excitation spectrum, emission and excitation maximum, fluorescence quantum yield and lifetime. It was found that the monoanion is the most fluorescent chromospheres (Phif=0.66, compared to Phif=0.25 for dianion, 0.18 for cation and 0.0 for the neutral form). Together with the computed pKas, we are able to suggest that the monoanion plays a major role under physiological conditions when DBFL is used as a fluorescence probe, contrary to the assumption in literature.