We present a method for the evaluation of fluorescence fluctuations on the basis of Mandel’s Q parameter, using sampling time-dependent factorial cumulants. By relating the Q parameter to the sampling time, we obtain the mean single molecule rate (mSMR), an easy to interpret expression that provides both brightness and diffusion information. The model is suitable for the widely used confocal setups with single photon excitation and a single detection channel. We present a way to correct the mSMR for afterpulsing, dead time and background noise. To account for photokinetic effects at short sampling times, we expand the model by a simple on/off isomerization term, which is similar to the well-known triplet model. The functionality of the mSMR is shown using Monte Carlo simulations. The correction mechanisms and the experimental applicability of the model are then demonstrated by DNA measurements of defined composition. By systematically analyzing DNA mixtures, we can show that at large sampling times, the mSMR correctly describes the single molecule brightness rates and the diffusive properties of DNA molecules. At short sampling times, the photokinetic effects of isomerization are accurately described by the mSMR model. Since additionally the mSMR can easily be corrected for measurement artefacts such as detector dead time, afterpulsing and background noise, this is a valuable advantage over the standard method of fluorescence correlation spectroscopy.

我们提出了一种基于曼德尔 Q 参数的荧光波动评估方法，使用采样时间相关的阶乘累积量。通过将 Q 参数与采样时间相关联，我们获得了平均单分子速率(mSMR)，一种易于解释的表达式，可提供亮度和扩散信息。该模型适用于具有单光子激发和单检测通道的广泛使用的共聚焦设置。我们提出了一种针对后脉冲、死区时间和背景噪声校正 mSMR 的方法。为了解释短采样时间的光动力学效应，我们通过一个简单的开/关异构化项扩展模型，这类似于众所周知的三重态模型。mSMR 的功能使用 Monte Carlo 模拟显示。然后通过定义成分的 DNA 测量来证明模型的校正机制和实验适用性。通过系统地分析 DNA 混合物，我们可以证明在大采样时间，mSMR 正确地描述了单分子亮度率和 DNA 分子的扩散特性。在较短的采样时间内，mSMR 模型准确描述了异构化的光动力学效应。此外，由于 mSMR 可以很容易地针对检测器死时间、后脉冲和背景噪声等测量假象进行校正，因此这是优于荧光相关光谱学标准方法的宝贵优势。