Hillesheim Lindsey N, Müller Joachim D
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biophys J. 2003 Sep;85(3):1948-58. doi: 10.1016/S0006-3495(03)74622-0.
Fluorescence fluctuation spectroscopy utilizes the signal fluctuations of single molecules for studying biological processes. Information about the biological system is extracted from the raw data by statistical methods such as used in fluctuation correlation spectroscopy or photon counting histogram (PCH) analysis. Since detectors are never ideal, it is crucial to understand the influence of photodetectors on signal statistics to correctly interpret the experimental data. Here we focus on the effects of afterpulsing and detector dead-time on PCH statistics. We determine the dead-time and afterpulse probability for our detectors experimentally and show that afterpulsing can be neglected for most experiments. Dead-time effects on the PCH are concentration-dependent and become significant when more than one molecule is present in the excitation volume. We develop a new PCH theory that includes dead-time effects and verify it experimentally. Additionally, we derive a simple analytical expression that accurately predicts the effect of dead-time on the molecular brightness. Corrections for non-ideal detector effects extend the useful concentration range of PCH experiments and are crucial for the interpretation of titration and dilution experiments.
荧光涨落光谱法利用单分子的信号涨落来研究生物过程。关于生物系统的信息通过诸如涨落相关光谱法或光子计数直方图(PCH)分析中所使用的统计方法从原始数据中提取。由于探测器并非理想状态,了解光电探测器对信号统计的影响对于正确解释实验数据至关重要。在此,我们重点关注后脉冲和探测器死时间对PCH统计的影响。我们通过实验确定了我们探测器的死时间和后脉冲概率,并表明在大多数实验中后脉冲可以忽略不计。死时间对PCH的影响取决于浓度,当激发体积中存在不止一个分子时会变得显著。我们开发了一种包含死时间影响的新PCH理论并通过实验进行了验证。此外,我们推导了一个简单的解析表达式,该表达式能准确预测死时间对分子亮度的影响。对非理想探测器效应的校正扩展了PCH实验的有用浓度范围,对于滴定和稀释实验的解释至关重要。