Abstract
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.
Original language | English (US) |
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Title of host publication | Ionotropic Glutamate Receptor Technologies |
Publisher | Springer New York |
Pages | 221-239 |
Number of pages | 19 |
ISBN (Electronic) | 9781493928125 |
ISBN (Print) | 9781493928118 |
DOIs | |
State | Published - Sep 25 2015 |
Keywords
- Digital imaging
- Fura-2
- Glutamate
- Hippocampal neuron
- Intracellular calcium concentration
- Ionotropic glutamate receptor
- Nmda receptor
- Single-cell electroporation