Ensemble and single-molecule fluorescence spectroscopy of a calcium-ion indicator dye.
The spectroscopic properties of Calcium Green 2 (CG-2), a dual-fluorophore Ca(2+) 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 Ca(2+) free solutions, CG-2 exists primarily as a highly quenched intramolecular dimer, but when bound to Ca(2+), 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 Förster coupling between fluorophores in conjunction with irreversible photodamage to one of the constituent chromophores.
Bagh
Sangram
S
Department of Chemistry, University Of Saskatchewan, SK, Canada.
Paige
Matthew F
MF
D000465:Algorithms; D002118:Calcium / Q000032:analysis* / Q000737:chemistry; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007477:Ions; D015394:Molecular Structure; D009930:Organic Chemicals / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007477:Ions; D009930:Organic Chemicals; C071336:calcium green; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium