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.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - 2000|
Garcia Parajo, M. F., Segers-Nolten, G. M. J., Veerman, J. A., Greve, J., & van Hulst, N. F. (2000). Real-time light-driven dynamics of the fluorescence emission in individual green fluorescent proteins. Proceedings of the National Academy of Sciences of the United States of America, 97(13), 7237-7242. https://doi.org/10.1073/pnas.97.13.7237