ON OFF BLINKING AND SWITCHING BEHAVIOR OF SINGLE MOLECULES OF GREEN FLUORESCENT PROTEIN/

Optical studies of individual molecules at low and room temperature ca n provide information about the dynamics of local environments in soli ds, liquids and biological systems unobscured by ensemble averaging(1- 14). Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jell fish Aequorea victoria. Wild-type GFP and its mutant have attracted in terest as fluorescent biological labels because the fluorophore may be formed in vivo(15,16). GFP mutants immobilized in aereated aqueous po lymer gels and excited by 488-nm light undergo repeated cycles of fluo rescent emission ('blinking') on a timescale of several seconds-behavi our that would be unobservable in bulk studies. Eventually the individ ual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic s witches or optical storage elements, addressable on the single-molecul e level.