Are intracellular ionic concentrations accessible using fluorescent probes? The example of Mag-indo-1

The study of the physicochemical properties of Mag-indo-1, a fluorescent probe used for intracellular magnesium measurements, has shown that in a biological environment the deprotonated form of this probe is in simultaneous equilibrium with a protonated form, a protein and a magnesium-bound form. The complex emission fluorescence spectrum emitted by a single living cell was analyzed using a computerized method, allowing the evaluation of the contribution of each species of the Mag-indo-1 to the cellular fluorescence. This approach used to evaluate intracellular Mg2+ concentration has also shown the variability of the important participation of protein-bound Mag-indo-1 to the cellular fluorescence. Thus the widely used ratioing method, unable to take into account this variability, cannot afford a reliable evaluation of [Mg2+]. Whatever the technique used for investigation (microfluorimetry, flow cytometry, etc.) the evaluation of [Mg2+]i using the fluorescent probe Mag-indo-1 requires a method able to quantify, in complex fluorescence, the fluorescence intensity of the forms involved in the equilibrium with Mg2+.