Confocal and multiphoton imaging of intracellular Ca(2+)

This chapter compares the imaging capabilities of a range of systems including multiphoton microscopy in regard to measurements of intracellular Ca(2+) within living cells. In particular, the excitation spectra of popular fluorescent Ca(2+) indicators are shown during 1P and 2P excitation. The strengths and limitations of the current indicators are discussed along with error analysis which highlights the value of matching the Ca(2+) affinity of the dye to a particular aspect of Ca(2+) signaling. Finally, the combined emission spectra of Ca(2+) and voltage sensitive dyes are compared to allow the choice of the optimum combination to allow simultaneous intracellular Ca(2+) and membrane voltage measurement.