Real-time light-driven dynamics of the fluorescence emission in individual green fluorescent proteins

M.F. Garcia Parajo, G.M.J. Segers-Nolten, J.A. Veerman, J. Greve, N.F. van Hulst

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143 Citations (Scopus)
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Abstract

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 μs 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.
Original languageEnglish
Pages (from-to)7237-7242
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number13
DOIs
Publication statusPublished - 2000

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