Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca²⁺ (K(d) ~20 μM, 6 μM and 12 μM respectively) and selectivity over Mg²⁺ (K(d) ~2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca²⁺ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn²⁺, Cd²⁺ and Gd³⁺. Interestingly, only in the case of BTC were spectral differences apparent between Ca²⁺ and other metal ions. For example, the presence of Zn²⁺ increased BTC fluorescence 6-fold at the Ca²⁺ isosbestic point, suggesting that this dye may be used as a fluorescent Zn²⁺ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca²⁺ indicator for most imaging applications. ETC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn²⁺. (C) 2000 Harcourt Publishers Ltd.