Calcium imaging to study NMDA receptor-mediated cellular responses

Measuring changes in intracellular Ca²⁺+ concentration ([Ca²⁺+]i) with optical methods is a useful approach to study the function and regulation of Ca²⁺+-permeable ion channels, such as ionotropic glutamate receptors. Here we describe a practical method for monitoring changes in [Ca²⁺+]i that employs the widely used Ca²⁺+ indicator, fura-2. Upon binding Ca²⁺+, the excitation maximum of fura-2 shifts from 380 to 340 nm. Therefore, the ratio of fura-2 fluorescence from cells excited at 340 relative to 380 nm tracks changes in [Ca²⁺+]i; importantly this ratio is independent of dye concentration, optical path length, or illumination intensity. We provide instructions for calibrating an imaging system and using ratio-metric analysis for processing fura-2 fluorescence images to represent [Ca²⁺+]i. Common technical problems are discussed in a section devoted to troubleshooting. Fura-2-based digital imaging has become a widely used technique with broad applicability. We describe methods to accomplish particularly common [Ca²⁺+]i imaging goals; however, these provide a versatile foundation that can be further developed into more complex approaches to acquire [Ca²⁺+]i-dependent images with higher temporal or spatial resolution, and from more challenging preparations.