Biomolecules and nanostructures
The Optical Sciences group studies the interaction of light and matter. Our current focus is on detection and sensing/imaging with an emphasis on the development of integrated photonics. We are part of Twente University's Department of Science and Technology and member of the MESA+ institute.
Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules
Garcia-Parajo MF, Segers-Nolten GMJ, Veerman JA, Greve J, van Hulst NF
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (PNAS)
vol 97 issue 13: p7237-p7242 JUN 20 2000
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 mu 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 approximate to 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 approximate to 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.