Siebrasse Jan Peter, Kubitscheck Ulrich
Institute for Physical and Theoretical Chemistry, Department of Biophysical Chemistry, University of Bonn, Bonn, Germany.
Methods Mol Biol. 2009;464:343-61. doi: 10.1007/978-1-60327-461-6_19.
Microscopic imaging of single fluorescent molecules within cells provides a molecular, real-time view of physiological processes in vivo. Single fluorescent molecules produce diffraction-limited light spots in the image plane, which can be localised with a very high precision. In single-molecule fluorescence microscopy (SMF) the achievable localisation precision depends only on the signal-to-noise ratio (SNR) and the stability of the optical setup. Typically values between 20 and 40 nm can be achieved. Highly dynamic processes and Brownian motion characterised by diffusion coefficients <20 microm(2)/sec can be followed by high-speed imaging, hence the method is an ideal tool to study intranuclear protein or ribonucleoprotein particle mobility. In contrast to conventional techniques, different forms of mobility in a heterogeneous system may well be distinguished from each other. Furthermore, specific binding and bimolecular interaction events can be followed at the single molecule level. A prominent example of an application is the study of nucleocytoplasmic transport one molecule at a time. In this case, the high localisation precision allows to analyse the binding site distribution of single molecules at the nuclear pore complex, and the high time resolution allows determination of the binding duration of soluble receptors and transport substrates.
细胞内单个荧光分子的显微成像提供了体内生理过程的分子实时视图。单个荧光分子在图像平面上产生受衍射限制的光斑，其可以以非常高的精度进行定位。在单分子荧光显微镜（SMF）中，可实现的定位精度仅取决于信噪比（SNR）和光学装置的稳定性。通常可以实现20至40纳米之间的值。通过高速成像可以跟踪具有<20微米²/秒扩散系数的高度动态过程和布朗运动，因此该方法是研究核内蛋白质或核糖核蛋白颗粒流动性的理想工具。与传统技术相比，异质系统中不同形式的流动性很可能彼此区分开来。此外，特定的结合和双分子相互作用事件可以在单分子水平上进行跟踪。一个突出的应用例子是一次研究一个分子的核质运输。在这种情况下，高定位精度允许分析单分子在核孔复合体处的结合位点分布，高时间分辨率允许确定可溶性受体和运输底物的结合持续时间。
