Niesner Raluca, Gericke Karl-Heinz
Institut für Physikalische und Theoretische Chemie, Technische Universität Braunschweig, Hans-Sommer Strasse 10, 38106 Braunschweig, Germany.
J Chem Phys. 2006 Apr 7;124(13):134704. doi: 10.1063/1.2179793.
Fluorescence fluctuation experiments are performed in single-molecule detection regime if the fluorescence of at most one molecule is registered at a time. Although the significance of such experiments for investigations of complex nonergodic systems like those met in the biosciences has been stressed out by many scientists, the quantitative and accurate determination of the single-molecule detection regime received rather little attention. In this work we present a method based on the photon counting histogram (PCH) analysis, which enables the determination of the average number N of molecules within the observation volume, for which only the fluorescence of individual molecules is detected at a time. Thus, the accurate design of fluorescence fluctuation experiments performed in single-molecule detection regime is possible. Demonstrative fluorescence fluctuation experiments based on two-photon excitation are performed on diluted solutions of coumarin 153, in order to verify the potential of the PCH analysis in experiments on the single-molecule detection level. If the mean number N of molecules within the excitation volume is larger than 0.048, the probability to simultaneously detect the fluorescence of two or more molecules is no longer negligible, i.e., no single-molecule detection regime. If the mean number N of molecules is lower than 0.0057, the detection limit of the method is reached, i.e., the fluorescence signal cannot be distinguished from the background. Consequently, the concentration of coumarin 153 characteristic for the single-molecule detection regime lies in the range 13-110 pmol/l for the given experimental conditions. We also investigate the influence of the molecular brightness, i.e., detected photons per fluorophore molecule and sampling time, on the single-molecule detection regime.
如果每次最多记录一个分子的荧光，那么荧光涨落实验是在单分子检测模式下进行的。尽管许多科学家都强调了此类实验对于研究生物科学中遇到的复杂非遍历系统的重要性，但单分子检测模式的定量和准确确定却很少受到关注。在这项工作中，我们提出了一种基于光子计数直方图（PCH）分析的方法，该方法能够确定观测体积内分子的平均数量N，在此情况下每次仅检测单个分子的荧光。因此，在单分子检测模式下进行的荧光涨落实验的精确设计成为可能。基于双光子激发进行了示范性荧光涨落实验，实验对象是香豆素153的稀释溶液，以验证PCH分析在单分子检测水平实验中的潜力。如果激发体积内分子的平均数N大于0.048，同时检测到两个或更多分子荧光的概率就不再可以忽略不计，即不存在单分子检测模式。如果分子的平均数N低于0.0057，就达到了该方法的检测极限，即荧光信号无法与背景区分开来。因此，在给定实验条件下，单分子检测模式所特有的香豆素153浓度范围为13 - 110 pmol/l。我们还研究了分子亮度（即每个荧光团分子检测到的光子数）和采样时间对单分子检测模式的影响。
