Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium 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. This cell-permeant Fluo-3FF is an analog of Fluo-3 AM with much lower calcium binding affinity, Kd ~10 µM. Like Fluo-3 AM, this indicator is essentially nonfluorescent, but exhibits strong fluorescence enhancement with no spectral shift upon entering cells and binding calcium..