Zhou Ruobo, Schlierf Michael, Ha Taekjip
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Methods Enzymol. 2010;475:405-26. doi: 10.1016/S0076-6879(10)75016-3.
During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. Yet these techniques would be even more powerful if they would be combined, that is, single-molecule manipulation with an orthogonal single-molecule observation. Here, we present a recently developed approach to combine single-molecule optical tweezers with single-molecule fluorescence spectroscopy. Optical tweezers are used to manipulate and observe mechanical properties on the nanometer scale and piconewton force range. However, once the force range is in the low piconewton range or less, the spatial resolution of optical tweezers decreases significantly. In combination with fluorescence spectroscopy, like Förster resonance energy transfer (FRET), we are able to observe nanometer fluctuations and internal conformational changes in a low-force regime. The possibility to place fluorescent labels at nearly any desired position and a sophisticated design of the experiment increases the amount of information that can be extracted in contrast to pure mechanical or fluorescence experiments.
在过去十年中，各种强大的单分子技术不断发展，并有助于解决生命科学中的重要问题。然而，如果将这些技术结合起来，即单分子操作与正交单分子观测相结合，它们将更加强大。在此，我们展示了一种最近开发的将单分子光镊与单分子荧光光谱相结合的方法。光镊用于在纳米尺度和皮牛顿力范围内操纵和观察机械性能。然而，一旦力范围处于低皮牛顿范围或更小，光镊的空间分辨率就会显著降低。与荧光光谱（如Förster共振能量转移（FRET））相结合，我们能够在低力状态下观察纳米级波动和内部构象变化。与纯机械或荧光实验相比，在几乎任何所需位置放置荧光标记的可能性以及实验的精巧设计增加了可提取的信息量。
