Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Fluo-3 and Rhod-2 are most commonly used among the visible light-excitable calcium indicators. Fluo-3 indicators are widely used in flow cytometry and confocal laser-scanning microscopy. More recently, Fluo-3, AM has been extensively used in cell-based high-throughput screening assays for functional GPCR assays. Fluo-3 is essentially nonfluorescent unless bound to Ca2+ and exhibits a quantum yield at saturating Ca2+ of ~0.14 and a Kd for Ca2+ of 390 nM.