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Experimental approach to extend the range for counting fluorescent molecules based on photon-antibunching

机译：实验方法扩展基于光子反聚束的荧光分子计数范围

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摘要

In single-molecule fluorescence spectroscopy photon-antibunching is frequently used to prove the occurrence of single fluorophores. Furthermore, the relative frequency of coincident photon pairs was also used to determine the number of fluorophores in the diffraction limited observation volume of a confocal microscope. However, the ability to count fluorophores is so far limited to ～ 3 molecules due to saturation of the calibration curve with increasing number of fluorophores. Recently, we introduced a novel theoretical framework for counting the number of emitting molecules by analyzing photon-distributions acquired with a confocal microscope using four single-photon detectors. Here, we present the experimental realization of the proposed scheme in a confocal setup using novel multi-channel photon-counting electronics and DNA constructs that were labelled with five fluorophores. Our experimental results give a clear correlation between the number of estimated fluorophores and the number of bleaching steps for DNA probes conjugated with five ATT0647N labels with an error of ～20%. Moreover, we could acquire experimental data for up to 15 fluorophores indicating the simultaneous occurrence of three DNA probes. Our experiments put into perspective that the analysis of photon-distributions acquired with four detection channels is suited to count the number of fluorescently labelled molecules in larger aggregates or clusters with potential for applications in molecular and cell biology and for time-resolved analysis of multi-chromophoric compounds in material sciences.
机译：在单分子荧光光谱中，经常使用光子反聚束来证明单个荧光团的发生。此外，重合光子对的相对频率还用于确定共聚焦显微镜的衍射受限观察体积中的荧光团数。但是，由于校准曲线随着荧光团数量的增加而饱和，因此荧光团的计数能力目前仅限于约3个分子。最近，我们引入了一种新颖的理论框架，用于通过分析使用四个单光子探测器的共聚焦显微镜获得的光子分布来计算发射分子的数量。在这里，我们介绍了使用新的多通道光子计数电子设备和被五个荧光团标记的DNA构建物在共聚焦设置中实验方案的实验实现。我们的实验结果给出了与5个ATT0647N标记缀合的DNA探针的估计荧光团数与漂白步骤数之间的明确相关性，其误差约为20％。此外，我们可以获得多达15个荧光团的实验数据，表明同时出现了3个DNA探针。我们的实验表明，通过四个检测通道获取的光子分布分析适合于计算较大的聚集体或簇中荧光标记的分子的数量，这些分子在分子和细胞生物学中的应用以及对多分子的时间分辨分析具有潜力。材料科学中的发色化合物。

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