Van Orden A, Machara N P, Goodwin P M, Keller R A
Chemical Science and Technology Division, MS M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545.
Anal Chem. 1998 Apr 1;70(7):1444-51. doi: 10.1021/ac970545k.
We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species: Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.
我们报告了一种多重技术，通过对单分子荧光猝发大小和猝发内荧光衰减率进行相关测量，来识别流动样本流中的单个荧光分子。在含有罗丹明6G和异硫氰酸四甲基罗丹明这两种荧光物质的稀释混合物的流动样本流中，对单个荧光分子同时测量了这些量。通过对我们实验的详细蒙特卡罗模拟，我们计算了识别这些物质的单分子检测效率和置信水平，并确定了单分子识别的主要误差来源。这里报道的技术适用于区分具有相似光谱特性的荧光团，并且只需要一个激发波长和一个荧光发射检测通道。
