Ha T, Enderle T, Ogletree D F, Chemla D S, Selvin P R, Weiss S
Molecular Design Institute, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6264-8. doi: 10.1073/pnas.93.13.6264.
We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.
通过测量单个供体荧光团与单个受体荧光团之间的能量转移，我们将荧光共振能量转移（FRET）的灵敏度扩展到了单分子水平。近场扫描光学显微镜（NSOM）用于获取由短DNA分子连接的供体和受体荧光团的同步双色图像及发射光谱。利用供体或受体的光破坏动力学来确定能量转移的存在及效率。用于测量荧光团集合体上能量转移的经典方程针对单分子测量进行了修正。与集合体测量不同，在单对FRET测量中可以观察到分子尺度上的动态事件，因为它们不会被随机平均所抵消。单对FRET可能实现对单个生物大分子内构象变化（如纳米尺度上的旋转和距离变化）的监测。
