Fluorescent Calcium Indicators:Subcellular Behavior and Use in Confocal Imaging

Most animal cells maintain a large gradient of free calcium across the plasma membrane (10,000-fold or more), with intracellular free calcium held at a level of about 100 nM . To accomplish this, cells have an array of molecular machinery including ATP-driven calcium pumps, calcium exchangers, intracellular calcium storage compartments, and an assortment of calcium binding proteins (1 –6 ). This machinery is needed because calcium is used ubiquitously as a signaling molecule, involving numerous receptors and a complex maze of intracellular signaling cascades (7 –10 ). One approach to the study of this biological arena is the tracking of calcium levels in different subcellular regions. Our goal is to consider how fluorescent calcium indicators, in conjunction with confocal microscopy, can best be used to image subcellular calcium dynamics.