Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy
Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy
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DOI：
10.1021/jp980965t
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发表时间：
1998-08-13
影响因子：
2.9
通讯作者：
Seidel, CAM
中科院分区：
文献类型：
--
作者：
Fries, JR;Brand, L;Seidel, CAM
利用共聚焦落射照明显微镜和脉冲激光，介绍了最近发展起来的实时光谱技术BIFL（猝发积分荧光寿命）的新应用。BIFL记录了关于测量的宏观时间尺度和荧光寿命的每个探测到的光子的两种不同类型的信息。因此，它被证明是非常适合通过其特征荧光寿命来识别自由扩散的单个染料分子。这允许对开放体积元件中的染料分子进行选择性计数，并开辟了使用最近推导的理论模型量化染料分子相对浓度的可能性，该模型分析了样本调查中获得的爆发尺寸分布。提出了一个封闭的理论来计算特定染料引起包含一定数量的检测光子的荧光猝发的概率。它考虑了激发辐照度在检测体积上的分布以及荧光和检测电子器件的饱和效应、通过检测体积的不同渡越时间的概率以及多分子事件的概率。采用BIFL结合选择性计数法，分别测定了罗丹明B和罗丹明6G两种染料在单独溶液和混合溶液中的浓度。所得到的结果是一致的，与所施加的染料浓度，并与荧光相关光谱（FCS）的同时测量。介绍的方法是一个适当的工具，在均相测定高度稀释的样品进行完整的表征和定量分析。
Using a confocal epi-illuminated microscope together with a pulsed laser, new applications of the recently developed, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) an introduced. BIFL registers two different types of information on every detected photon with regard to the macroscopic time scale of a measurement and to the fluorescence lifetime. Thus, it is shown to be well suited to identify freely diffusing single dye molecules via their characteristic fluorescence lifetime. This allows for selective counting of dye molecules in an open volume element and opens up the possibility to quantify the relative concentration of the dye molecules, using a recently derived theoretical model, which analyzes the obtained burst size distribution of a sample survey. A closed theory is presented to calculate the probability of a specific dye to cause a fluorescence burst containing a certain number of detected photons. It considers the distribution of the excitation irradiance over the detection volume together with saturation effects of the fluorescence and of the detection electronics, the probability of different transit times through the detection volume, and the probability of multimolecule events. Using BIFL together with selective counting, the concentration of two dyes, Rhodamine B and Rhodamine 6G, in separate solutions and in a mixture were determined. The obtained results are consistent with the applied dye concentrations and with simultaneous measurements by fluorescence correlation spectroscopy (FCS). The introduced method is an appropriate tool for the complete characterization and quantitative analysis of a highly diluted sample in homogeneous assays.