Földes-Papp Zeno, Baumann Gerd, Demel Ulrike, Tilz Gernot P
Clinical Immunology and Jean Dausset Laboratory, Graz University Medical School and Hospital, Auenbruggerplatz 8, A-8036 Graz, LKH, Austria.
Curr Pharm Biotechnol. 2004 Apr;5(2):163-72. doi: 10.2174/1389201043376986.
Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.
许多分子相互作用、生化和化学反应的理论模型是在单分子水平上描述的，尽管我们对生化/化学结构和动力学的了解主要源于对多分子系统的研究。目前，有四个实验平台可用于观察单个荧光分子的运动和行为：宽场落射照明、近场光学扫描、激光扫描共聚焦显微镜和多光子显微镜。这些平台与诸如荧光共振能量转移（FRET）、荧光自相关或双色交叉相关光谱（FCS）、荧光偏振各向异性、荧光猝灭和荧光寿命测量等分析方法相结合。本文的原创贡献集中于对在单相（如溶液或膜）中自由扩散的单个分子进行计数和表征，且不存在流体动力流、固定化或强度轨迹的爆发大小分析。例如，如本文原始文章所示，这可以通过荧光自相关或双色交叉相关光谱来实现。文中讨论了三个标准（Földes-Papp（2002年）《蝶啶》，第13卷，第73 - 82页；Földes-Papp等人（2004年a）《免疫学方法杂志》，第286卷，第1 - 11页；Földes-Papp等人（2004年b）《免疫学方法杂志》，第286卷，第13 - 20页），用于对在溶液或膜中自由扩散的同一个单个（个体）分子进行从亚毫秒到数小时的连续测量。为单分子荧光检测开发的“算法”被称为“同一单荧光分子模式”。所发现结果的一个有趣应用是在免疫学领域。该理论在实验结果中的应用表明该理论与实验结果一致。本文全面介绍了关于单（溶液）相单分子荧光自相关或双色交叉相关光谱（SPSM-FCS）的新观点。随着技术不断改进，对FCS/FCCS的要求所涉及的极限也相应地被突破。
