Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules.
Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the "on-off" blinking observed in the Ser-65 --> Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 micros and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on- and off-times renders a characteristic off-time of 1.6 +/- 0.2 s and allows us to calculate a transition yield of approximately 0.5 x 10(-5) from the emissive to the nonemissive state. The saturation excitation intensity at which on- and off-times are equal is approximately 1.5 kW/cm(2). On the basis of the single-molecule data we calculate an absorption cross section of 6.5 x 10(-17) cm(2) for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level.
Garcia-Parajo
M F
MF
Applied Optics Group and Biophysical Techniques, Department of Applied Physics and MESA(+) Research Institute, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands. m.f.garciaparajo@tn.utwente.nl
Segers-Nolten
G M
GM
Veerman
J A
JA
Greve
J
J
van Hulst
N F
NF
D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins