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Real-Time Light-Driven Dynamics of the Fluorescence Emission in Single Green Fluorescent Protein Molecules
M. F. Garcia-Parajo, G. M. J. Segers-Nolten, J.-A. Veerman, J. Greve and N. F. Van Hulst
Proceedings of the National Academy of Sciences of the United States of America
Vol. 97, No. 13 (Jun. 20, 2000), pp. 7237-7242
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/122797
Page Count: 6
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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 μ s 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 ≈ 0.5 × 10-5 from the emissive to the nonemissive state. The saturation excitation intensity at which on- and off-times are equal is ≈ 1.5 kW/cm2. On the basis of the single-molecule data we calculate an absorption cross section of 6.5 × 10-17 cm2 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.
Proceedings of the National Academy of Sciences of the United States of America © 2000 National Academy of Sciences