<title>Confocal fluorescence lifetime imaging of ion concentrations</title>

Ion concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for specific ions and exhibit marked changes in their photophysical properties upon binding ions. The fluorescence decay behavior of the probes in the presence of ions can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within living cells. Here we describe the fluorescence lifetime properties of the free calcium probe CalciumGreen and the pH probe carboxy SNAFL-1. The potential of fluorescence lifetime imaging is illustrated by the imaging of Ca²⁺ concentrations and pH in single cells. In the case of the emission ratio probe c.SNAFL-1, it was possible to determine the pH in the same cell using both the ratio and the fluorescence lifetime method. It turns out that no cumbersome in vivo calibration procedure is required when c.SNAFL-1 is used for quantitative fluorescence lifetime imaging of pH in single cells.