Using two dyes with the same fluorophore to monitor cellular calcium concentration in an extended range.

We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications. We provide the exact analytic expression relating measured fluorescence to [Ca(2+)] at equilibrium and an approximate analytic expression that does not require the equilibrium assumption. The sensitivity of the method is calculated numerically for two useful dye pairs. As illustrative application of the enhanced measurement, we use fluo-4 and fluo-4FF to image the calcium wave produced by a cardiac myocyte in response to a small artificial calcium spark.