An Ensemble and Single-molecule Fluorescence Spectroscopy Investigation of Calcium Green 1, a Calcium-ion Sensor

The calcium-ion indicator dye, Calcium Green 1 (CG-1), has been characterized using a combination of ensemble and single-molecule optical spectroscopy measurements. In terms of ensemble measurements, CG-1 demonstrated a strong increase in fluorescence emission as a function of increasing [Ca²⁺]. This was accompanied by a change in the relative proportions of two chemical forms of the dye, each with a different fluorescence lifetime, which were found to co-exist in solution. From single-molecule fluorescence measurements, it was found that the fluorescence intensity and photobleaching time (on-time) of each CG-1 molecule was invariant with [Ca²⁺] and that changes in ensemble fluorescence intensity simply correlates with the number of fluorescent molecules in solution. These results are compared with that of the related system, Calcium Green 2 (CG-2), and the mechanisms of operation of these two indicator dyes are discussed.