Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons
Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons
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DOI：
10.1021/ac025730z
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发表时间：
2002-10-15
影响因子：
7.4
通讯作者：
Sauer, M
中科院分区：
文献类型：
--
作者：
Weston, KD;Dyck, M;Sauer, M
描述并演示了一种简单的新方法，用于测量独立发射器的数量以及荧光强度，寿命和发射波长，用于单分子和多色系统的轨迹和图像，使用单个PC插件卡进行时间相关单光子计数。探测体积中存在的独立发射体的数量可以用类似于经典反聚束实验的方式利用光子间时间来确定。与传统的基于脉冲激光激发和使用时间-振幅转换器直接测量同步光子对的符合分析不同，光子间距离是通过在两个光谱分离的探测器上以纳秒时间分辨率记录每个光子的绝对到达时间来获取的。由光物理参数波动引起的强度变化可与单发色团和多发色团强度轨迹中发射体数量变化（例如光漂白）引起的强度变化区分开来。这是第一份证明基于检测体积内独立发射物种数量的对比度成像的报告。
A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.