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bin时间对单光子激发的光子计数直方图的影响。

Effect of bin time on the photon counting histogram for one-photon excitation.

作者信息

Perroud Thomas D, Huang Bo, Zare Richard N

机构信息

Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.

出版信息

Chemphyschem. 2005 May;6(5):905-12. doi: 10.1002/cphc.200400547.

Abstract

We have demonstrated that our photon counting histogram (PCH) model with the correction for one-photon excitation is valid at multiple bin times. The fitted apparent brightness and concentration follow the three-dimensional diffusion model. More importantly, the semi-empirical parameter, F, introduced in the PCH model for one-photon excitation to correct for the non-Gaussian shape of the observation volume, shows small variations with different bin times. These variations are consistent with the physical interpretation of F, and they do not affect the resolving power of the PCH model for one-photon excitation. Based on these findings, we extend the time-independent PCH analysis to time-dependent photon counting multiple histograms (PCMH). This model considers the effect of bin time on the PCH parameters in a way that is similar to fluorescence intensity multiple distribution analysis (FIMDA). From the same set of data, PCMH extracts time-dependent parameters (diffusion time and triplet-state relaxation time) as well as time-independent parameters (true specific brightness and true average number of molecules). Given a three- to fourfold experimental difference in molecular brightness, we find that PCMH can resolve each species in a two-species sample and extract their respective diffusion times even when fluorescence correlation spectroscopy cannot.

摘要

我们已经证明,我们的经过单光子激发校正的光子计数直方图(PCH)模型在多个箱时间下都是有效的。拟合得到的表观亮度和浓度遵循三维扩散模型。更重要的是,在PCH模型中引入的用于单光子激发以校正观测体积非高斯形状的半经验参数F,在不同的箱时间下变化很小。这些变化与F的物理解释一致,并且它们不会影响单光子激发的PCH模型的分辨能力。基于这些发现,我们将与时间无关的PCH分析扩展到与时间相关的光子计数多重直方图(PCMH)。该模型以类似于荧光强度多重分布分析(FIMDA)的方式考虑箱时间对PCH参数的影响。从同一组数据中,PCMH可以提取与时间相关的参数(扩散时间和三重态弛豫时间)以及与时间无关的参数(真实比亮度和真实平均分子数)。在分子亮度存在三到四倍的实验差异的情况下,我们发现即使荧光相关光谱法无法做到,PCMH也能分辨双物种样品中的每种物种并提取它们各自的扩散时间。

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