Palmer, A.E. and Tsien, R.Y.
Measuring calcium signaling using genetically targetable fluorescent indicators.  Nature Protocols 1: 1057-65 (2006).

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 B1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take ~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 ~1 d depending on the extent of analysis