Photophysics of Calcium Green 1 in vitro and in live cells

The absolute measurement of calcium activity in cells is hindered by the pr esence of different forms of the probe (adsorbed, protonated). Time-resolve d fluorescence spectroscopy can be a sensitive technique to separate contam inating forms of the probe. The fluorescent probe Calcium Green 1 was chara cterised, in vitro, in terms of calcium and also pH dependence, albumin ads orption, extent of hydrolysis, viscosity and medium polarity. A 1:1 stoichi ometry was obtained for the calcium complexation with a K-d value that agre es with reported values. A pK(a) of the BAPTA moiety was measured as 6.7. T he fluorescence lifetimes are slightly dependent on polarity but not on vis cosity. It was observed that most of the interfering forms of the probe sha re the common lifetimes of 0.5 and 3.5 ns. An alternative mechanism for Cal cium Green 1 sensing is proposed based on a ground state conformational equ ilibrium affecting the photoinduced electron transfer.