Calcium imaging to study NMDA receptor-mediated cellular responses

Kelly A Krogh, Stanley A. Thayer

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations


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 languageEnglish (US)
Title of host publicationIonotropic Glutamate Receptor Technologies
PublisherSpringer New York
Number of pages19
ISBN (Electronic)9781493928125
ISBN (Print)9781493928118
StatePublished - Sep 25 2015


  • Digital imaging
  • Fura-2
  • Glutamate
  • Hippocampal neuron
  • Intracellular calcium concentration
  • Ionotropic glutamate receptor
  • Nmda receptor
  • Single-cell electroporation


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