Characterizing multiple molecular states in single-molecule multiparameter fluorescence detection by probability distribution analysis
Characterizing multiple molecular states in single-molecule multiparameter fluorescence detection by probability distribution analysis
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DOI：
10.1021/jp711942q
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发表时间：
2008-07-17
影响因子：
3.3
通讯作者：
Seidel, Claus A. M.
中科院分区：
文献类型：
--
作者：
Kalinin, Stanislav;Felekyan, Suren;Seidel, Claus A. M.
概率分布分析（PDA）[M. Antonik等人，J. Phys. Chem. B 2006，110，6970]允许定量分析在福斯特共振能量转移（FRET）或荧光偏振实验中获得的单分子（SM）数据。通过明确考虑背景和散粒噪声的贡献，PDA准确地预测了各种参数，如FRET效率或荧光各向异性的一维直方图的形状。为了描述复杂的实验SM-FRET或偏振数据的系统组成的多个非相互转换的荧光状态，PDA理论的几个扩展。亮度变化和多分子事件的影响被认为是独立的检测体积参数，仅使用整体实验信号强度分布。扩展的PDA理论现在可以应用于分析任何混合物，通过使用任何先验模型或基于最大熵方法（MEM）的无模型反卷积方法。分析的准确性和自由参数的数量仅受数据质量的限制。校正PDA模型函数的多分子事件的存在允许在高SM浓度下测量，以避免由于非常长的测量时间而产生的伪影。已经开发了MEM和组合平均供体荧光寿命分析等工具，以区分PDA直方图的额外增宽是否可归因于结构异质性或染料伪影。以这种方式，实现了在几埃范围内的FRET实验中的最终分辨率，这允许分子埃光学区分一组固定距离和距离分布。通过仿真和实验数据的分析验证了扩展理论的正确性。
Probability distribution analysis (PDA) [M. Antonik et al., J. Phys. Chem. B 2006, 110, 6970] allows one to quantitatively analyze single-molecule (SM) data obtained in Forster resonance energy transfer (FRET) or fluorescence polarization experiments. By taking explicitly background and shot noise contributions into account, PDA accurately predicts the shape of one-dimensional histograms of various parameters, such as FRET efficiency or fluorescence anisotropy. In order to describe complex experimental SM-FRET or polarization data obtained for systems consisting of multiple non-interconverting fluorescent states, several extensions to the PDA theory are presented. Effects of brightness variations and multiple-molecule events are considered independently of the detection volume parameters by using only the overall experimental signal intensity distribution. The extended PDA theory can now be applied to analyze any mixture, by using any a priori model or a model-free deconvolution approach based on the maximum entropy method (MEM). The accuracy of the analysis and the number of free parameters are limited only by data quality. Correction of the PDA model function for the presence of multiple-molecule events allows one to measure at high SM concentrations to avoid artifacts due to a very long measurement time. Tools such as MEM and combined mean donor fluorescence lifetime analysis have been developed to distinguish whether extra broadening of PDA histograms could be attributed to structural heterogeneities or dye artifacts. In this way, an ultimate resolution in FRET experiments in the range of a few Angstrom is achieved which allows for molecular Angstrom optics distinguishing between a set of fixed distances and a distribution of distances. The extended theory is verified by analyzing simulations and experimental data.