Journal of Physical Chemistry A, Vol.110, No.22, 7057-7066, 2006
Ensemble and single-molecule fluorescence spectroscopy of a calcium-ion indicator dye
The spectroscopic properties of Calcium Green 2 (CG-2), a dual-fluorophore Ca2+ indicator dye, were characterized by a combination of steady state and time-resolved ensemble spectroscopic measurements, molecular mechanics calculations and single-molecule fluorescence spectroscopy. It was found that in Ca2+ free solutions, CG-2 exists primarily as a highly quenched intramolecular dimer, but when bound to Ca2+, the molecule adopts an extended, fluorescent conformation. The difference in emission properties of these two CG-2 conformations is explained in terms of simple exciton theory. Through single-molecule fluorescence measurements, we have shown that the bulk increase in ensemble fluorescence intensity correlates with a simple statistical increase in the number of fluorescent molecules in solution. In addition, we have also observed that the majority of CG-2 molecules photobleach in a single step, despite the molecule possessing two distinct fluorophores. A small fraction of molecules photobleach in multiple steps or show a series of transitions between emissive and nonemissive fluorescent states ("blinking"). We rationalize these photophysical phenomena using a simple model based on dipole-dipole Forster coupling between fluorophores in conjunction with irreversible photodamage to one of the constituent chromophores.