Measuring calcium signaling using genetically targetable fluorescent indicators

Nat Protoc. 2006;1(3):1057-65. doi: 10.1038/nprot.2006.172.


Genetically encoded Ca2+ indicators allow researchers to quantitatively measure Ca2+ dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca2+ in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca2+-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca2+ concentrations. Additionally, we highlight some of the challenges in observing organellar Ca2+ and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca2+ indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take approximately 1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take approximately 3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take approximately 1 d depending on the extent of analysis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers / analysis*
  • Biomarkers / metabolism
  • Calcium / analysis*
  • Calcium / metabolism
  • Calmodulin / metabolism
  • Calmodulin-Binding Proteins / metabolism
  • Cells, Cultured
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / metabolism*
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods


  • Biomarkers
  • Calmodulin
  • Calmodulin-Binding Proteins
  • Fluorescent Dyes
  • Calcium