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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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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proteins
fluorescence
blinking
excitation
statistical analysis
absorption cross sections
molecules
near fields
microscopy
intervals
saturation
scanning

Cite this

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title = "Real-time light-driven dynamics of the fluorescence emission in individual green fluorescent proteins",
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.",
author = "{Garcia Parajo}, M.F. and G.M.J. Segers-Nolten and J.A. Veerman and J. Greve and {van Hulst}, N.F.",
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doi = "10.1073/pnas.97.13.7237",
language = "English",
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Real-time light-driven dynamics of the fluorescence emission in individual green fluorescent proteins. / Garcia Parajo, M.F.; Segers-Nolten, G.M.J.; Veerman, J.A.; Greve, J.; van Hulst, N.F.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 13, 2000, p. 7237-7242.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Real-time light-driven dynamics of the fluorescence emission in individual green fluorescent proteins

AU - Garcia Parajo, M.F.

AU - Segers-Nolten, G.M.J.

AU - Veerman, J.A.

AU - Greve, J.

AU - van Hulst, N.F.

PY - 2000

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N2 - 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.

AB - 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.

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