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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.

Quantitative Measurement of Ca2+ and Zn2+ in Mammalian Cells Using Genetically Encoded Fluorescent Biosensors