Photophysics of Calcium Green 1 invitro and in live cells

The absolute measurement of calcium activity in cells is hindered by the presence of different forms of the probe (adsorbed, protonated). Time-resolved fluorescence spectroscopy can be a sensitive technique to separate contaminating forms of the probe. The fluorescent probe Calcium Green 1 was characterised, invitro, in terms of calcium and also pH dependence, albumin adsorption, extent of hydrolysis, viscosity and medium polarity. A 1:1 stoichiometry was obtained for the calcium complexation with a Kd value that agrees with reported values. A pKa of the BAPTA moiety was measured as 6.7. The fluorescence lifetimes are slightly dependent on polarity but not on viscosity. It was observed that most of the interfering forms of the probe share the common lifetimes of 0.5 and 3.5 ns. An alternative mechanism for Calcium Green 1 sensing is proposed based on a ground state conformational equilibrium affecting the photoinduced electron transfer.