Quantitative measurement of Ca2+ and Zn2+ in mammalian cells using genetically encoded fluorescent biosensors

Genetically encoded, ratiometric, fluorescent biosensors can be used to quantitatively measure intracellular ion concentrations in living cells. We describe important factors to consider when selecting a Ca(2+) or Zn(2+) biosensor, such as the sensor's dissociation constant (K(d')) and its dynamic range. We also discuss the limits of quantitative measurement using these sensors and reasons why a sensor may perform differently in different biological systems or subcellular compartments. We outline protocols for (1) quickly confirming sensor functionality in a new biological system, (2) calibrating a sensor to convert a sensor's FRET ratio to ion concentration, and (3) titrating a sensor in living cells to obtain its K(d') under different experimental conditions.