The design and properties of a series of calcium indicators which shift from rhodamine-like to fluorescein-like fluorescence on binding calcium

The prototype for a new series of ratio-mode fluorescence indicators of cytosolic free Ca2+ concentration ([Ca2+]i) has been developed. The fluorophore, termed FluoRhod, is a hybrid containing elements of the closely related fluorescein and rhodamine structures. The novel feature of the prototype indicator, FluoRhod-2, which incorporates a tetracarboxylate chelating element similar to that of 1,2-bis[2-aminophenoxy]ethane-N,N,N′,N′-tetraacetic acid (BAPTA), is that it shifts from rhodamine- to fluorescein-like fluorescence on binding Ca2+. The synthesis and properties of a series of tetracarboxylate derivatives of the FluoRhod fluorophore are described which led to the FluoRhod-2 structure with an apparent dissociation constant for Ca2+ and pKaadjusted to meet the requirements for a [Ca2+]i indicator in the ratio mode. The excitation and emission maxima of FluoRhod-2 are 537 nm and 566 nm in the absence of Ca2+ and 480 nm and 537 nm in the presence of Ca2+. The indicator can be used in either the dual excitation or dual emission mode or the ratio of these two ratios can be used by making dual excitation and dual emission measurements. The brightness of FluoRhod-2 is comparable to that of fura-2 with the advantage of excitation in the visible range. FluoRhod-2 is insensitive to pH from 6.5 to 7.5 when used in the ratio mode, irrespective of the extent to which the indicator is complexed to Ca2+.