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.
- Digital imaging
- Hippocampal neuron
- Intracellular calcium concentration
- Ionotropic glutamate receptor
- Nmda receptor
- Single-cell electroporation