Indo-1 Calcium Indicator
The ability to make ratio measurements with indo-1, and their derivatives is an important property of these probes. At low concentrations of the indicator, use of the 405/485 nm emission ratio for indo-1 allows accurate measurements of the intracellular Ca2+ concentration. Measurement by ratio analysis 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 indo-1 fluorescence can usually be made over a period of an hour without significant loss of fluorescence resulting from either leakage or bleaching. In addition, indo-1 is bright enough to permit measurements at intracellular concentrations of dye unlikely to cause significant Ca2+ buffering or damping of Ca2+ transients.
In contrast to fura indicators, which exhibit large changes in absorption on Ca2+ binding, the emission of indo-1 shifts from about 475 nm without Ca2+ to about 400 nm with Ca2+ when excited at about 350 nm (Figure 1). Indo-1 is especially useful for flow cytometry where it is easier to change the emission filters with a single excitation source (often the ultraviolet lines of the argon-ion laser in flow cytometers), and is particularly suited for multicolor fluorescence applications. Measurements with indo-5F can be carried out using the same instrument configurations as for indo-1.
|Figure 1: Fluorescence emission spectra of indo-1 (I1202) in solutions containing 0–39.8 µM free Ca2+.|