Clarke Richard W, Orte Angel, Klenerman David
Department of Chemistry, Cambridge University, Lensfield Road, Cambridge, CB2 1EW, UK.
Anal Chem. 2007 Apr 1;79(7):2771-7. doi: 10.1021/ac062188w. Epub 2007 Mar 8.
Two-color coincidence detection is a single-molecule fluorescence technique that is capable of resolving subpopulations of biomolecular complexes at very low concentrations. In this paper, we have developed a method that automatically determines appropriate thresholds for the analysis of sets of two-color coincidence data. This has the distinct advantage of allowing the rapid determination of optimized thresholds in a reproducible fashion. The trade-offs involved in such selections are that the thresholds should be high enough both to exceed the background photon count rates and to ensure a low rate of chance coincident events and that they should be low enough to give reasonably high rates of fluorescence events. Previously, thresholds were selected by judgment to balance these various separate considerations. The method reported in this paper incorporates the three factors into the maximization of a single value derived from the data as a function of the thresholds used in the two channels. The value that is maximized is a ratio of event rates, specifically the rate of coincident events above that expected by chance, divided by the total event rate; this is called the association quotient. In this paper, we demonstrate that maximization of the association quotient selects appropriate thresholds for data derived from dual-labeled duplex DNA samples over a range of concentrations and laser powers. This method should allow the application of two-color coincidence detection to more complex biological systems and cells where the sample concentration and background levels are more variable and where it is not possible to run separate control experiments to determine the latter statistics.
双色符合检测是一种单分子荧光技术，能够在极低浓度下分辨生物分子复合物的亚群。在本文中，我们开发了一种方法，可自动确定用于分析双色符合数据集的合适阈值。这具有显著优势，即能够以可重复的方式快速确定优化阈值。此类选择所涉及的权衡在于，阈值应足够高，既能超过背景光子计数率，又能确保偶然符合事件的发生率较低；同时，阈值应足够低，以产生合理的高荧光事件发生率。以前，阈值是通过判断来选择的，以平衡这些不同的单独考虑因素。本文报道的方法将这三个因素纳入一个从数据中导出的单一值的最大化过程中，该值是两个通道中使用的阈值的函数。被最大化的值是事件发生率的比率，具体而言，是偶然预期之上的符合事件发生率除以总事件发生率；这被称为关联商。在本文中，我们证明，关联商的最大化能为来自不同浓度和激光功率的双标记双链DNA样本的数据选择合适的阈值。该方法应能使双色符合检测应用于更复杂的生物系统和细胞，在这些系统和细胞中，样本浓度和背景水平变化更大，且无法进行单独的对照实验来确定后者的统计数据。
