Biomolecules and nanostructures
The Optical Sciences group studies the interaction of light and matter at the nanoscale.
We do this by exploring ways to shape light and its environment. It's what we call
active and passive control. Our current focus is on the interaction of light with
biomolecules and nanostructures. We are part of Twente
University's Department of Science and Technology and member of the
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.