Fura-2 is a ratiometric and sensitive indicator dye for measuring intracellular calcium. Since its introduction in 1985, fura-2 has been cited in thousands of papers that describe its applications in a wide variety of cells. These important Ca2+ indicators, developed by Roger Tsien and collaborators, have made a major contribution to advances in the understanding of the role of calcium in cellular regulation. The ability to make ratio measurements with fura-2 is an important property of this probe.
At low concentrations of the indicator, 340/380 nm excitation ratio for fura-2 allows accurate measurements of the intracellular Ca2+ concentration (Figure 1). Measuring by ratio considerably reduces the effects of uneven dye loading, leakage of dye, and photobleaching, as well as problems associated with measuring Ca2+ in cells of unequal thickness. Measurements of fura-2 fluorescence can usually be made over a period of an hour without significant loss of fluorescence resulting from either leakage or bleaching. In addition, fura-2 is bright enough to permit measurements at intracellular concentrations of dye unlikely to cause significant Ca2+ buffering or damping of Ca2+ transients.