Instrument Development for Imaging and Manipulation of Single Biomolecules

伊利诺伊大学厄巴纳-香槟分校（University of Illinois, Urbana-Champaign）已经获得了一笔拨款。Paul Selvin和Taekjip Ha开发新型单分子显微镜，这种显微镜结合了在模拟细胞自然环境的条件下观察和操纵单个生物分子的能力。这些分子被荧光染料或激发后发光的染料标记。可以通过监测这种荧光来收集有关分子位置和/或形状的信息。与此同时，这些分子受到各种环境实际压力——包括机械力和扭矩、电力和化学力以及温度变化。然后可以监测分子对这些压力的反应。具体来说，Selvin和Ha建议建立两个显微镜站。我们将以非常高的空间和时间分辨率一次检查一个分子。这使用了所谓的共聚焦光学，在操纵的同时，用强烈的、紧密聚焦的激光束对单个分子进行研究。另一种显微镜将同时观察许多单个分子，方法是将分子排列成阵列，并与漫射聚焦的光平行观察。这种方法可以更快地获得统计数据。这种平行检测对于监测不可逆反应尤为重要。高分辨率成像也将在这个显微镜站发展。这些显微镜将被用来检查各种特定的生物系统。一种是一类叫做分子马达的蛋白质。这些生物分子利用富含能量的小分子（如三磷酸腺苷，ATP）的化学能，并将其转化为机械（或其他形式）的能量。膜蛋白是另一类对生命至关重要的生物分子，但在许多情况下人们对其知之甚少。Selvin和Ha将研究几种离子通道（使细胞能够控制其盐（离子）组成的膜蛋白）如何根据小分子（配体）和跨膜电压打开和关闭。蛋白质、DNA和RNA的折叠反应对生命也是必不可少的。这些将通过通过温度或化学浓度的快速变化诱导折叠来研究。单分子研究彻底改变了我们对生物分子如何工作的理解。一次观察一个分子的能力不仅揭示了在集合测量中检测到的平均性质，而且可以得出相关性质的整个分布。单分子研究还揭示了生物化学反应的时间演化，如果是异步的，则无法通过集合测量观察到。与此同时，荧光技术的使用也在不断发生变革，通过对生物分子的某些部分进行特定标记，使科学家能够解剖生物分子的每个部分是如何移动的。单分子操作和荧光技术的结合有可能彻底改变我们对这些微小生物分子如何工作的理解。

A grant has been awarded to the University of Illinois, Urbana-Champaign, under the direction of Drs. Paul Selvin and Taekjip Ha to develop novel single molecule microscopes that combine the ability to both watch and manipulate individual biomolecules under conditions that simulate the native environment in a cell. The molecules are tagged with a fluorescent dye or dyes that emit light after excitation. Information about the position and/or shape of the molecule can be gathered by monitoring this fluorescence. At the same time, the molecules are put under a variety of environmentally realistic stresses - including mechanical forces and torques, electrical and chemical forces, and temperature changes. How the molecules react to these stresses can then be monitored. Specifically, Selvin and Ha propose to build two microscope stations. One will examine one molecule at a time with very high spatial and temporal resolution. This uses what is called confocal optics, where a single molecule is interrogated with an intense, tightly focussed laser beam, while being manipulated. The other microscope will look at many individual molecules simultaneously, by placing the molecules in an array and interrogating them in parallel with more diffusely focused light. This method can acquire statistical data much more rapidly. Such pParallel detection is particularly important for monitoring irreversible reactions. High-resolution imaging will also be developed on this microscope station. These microscopes will be used to examine a variety of specific biological systems. One is a class of proteins called molecular motors. These biomolecules uses the chemical energy of small energy-rich molecules (such as adenosine triphosphate, ATP) and convert it into mechanical (or other forms) of energy. Membrane proteins are another class of biomolecules central to life yet in many cases poorly understood. Selvin and Ha will study how several classes of ion channels - membrane proteins that enable the cell to control its salt (ion) composition - open and close in response to small molecules (ligand) and transmembrane voltage. Folding reactions of proteins, DNA, and RNA are also essential to life. These will be studied by inducing folding via rapid changes in temperature or chemical concentrations.Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties. Single molecule studies also reveal the time evolution of biochemical reactions, which, if asynchronous, are not observable via ensemble measurements. At the same time, there is an ongoing revolution in the use of fluorescence, which, via specific labeling to parts of the biomolecules, enables scientists to dissect how each part of the biomolecule is moving. The combination of single molecule manipulation and fluorescence techniques has the potential for revolutionizing our understanding of how these tiny biomolecules work.