Fluorescence lifetime microscopy of the sodium indicator sodium-binding benzofuran isophthalate in HeLa cells

The behavior of the sodium indicator sodium-binding benzofuran isophthalate (SBFI) is investigated in HeLa cells by time-resolved fluorescence microscopy. The fluorescence relaxation of SBFI in HeLa cells can be described by a triexponential for intracellular sodium concentration ([Na+](i)) between 0 and 90 mM. Changes in [Na+](i) affect neither the fluorescence relaxation times (0.21, 0.60, and 2.7 ns) nor the average decay time (2.2 ns). The preexponential factor of the shortest decay time is negative. However, the ratio of the fluorescence excitation signal at 340 nm to that at 380 nm increases with [Na+](i). To elucidate the behavior of SBFI in cells, experiments are performed on SBFI in buffer at various concentrations of sodium, potassium, and bovine serum albumin (BSA) and at various viscosities. The fluorescence decay is triexponential only in the presence of BSA. The relaxation times are independent of [Na+] and [BSA]. The preexponential factor of the shortest decay time is negative from a certain [BSA] on, which depends on [Na+]. The data indicate that interactions with intracellular components rather than microviscosity influence the SBFI behavior in cells. A model is suggested in which the fluorescence intensities are mainly determined by the signals from the Na(+)subset of SBFI and SBFI subset of protein complexes. (C) 2000 Academic Press.