Yutaka Sano, Yuji Itoh, Supawich Kamonprasertsuk, Leo Suzuki, Atsuhito Fukasawa, Hiroyuki Oikawa* and Satoshi Takahashi*,
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引用次数: 0
摘要
基于双色荧光检测的纳秒分辨荧光相关光谱法(ns-FCS)是一种强大的策略,可用于研究用供体和受体荧光团标记的生物大分子的快速动力学。ns-FCS 的标准方法使用两个单光子雪崩二极管(SPAD)检测单色信号(使用四个 SPAD 检测双色信号),以消除 SPAD 的后脉冲伪影,但却牺牲了利用光子数据计算相关图的效率。在此,我们证明了混合光电探测器(HPD)能够在 ns-FCS 中记录荧光光子,其基础是使用两个 HPD 检测双色信号的最小系统。然而,HPD 显示的后脉冲相对于光电检测率的产率(<10-4)远低于 SPAD(∼10-2),这仍然会妨碍相关测量。我们证明,简单的减法过程就能消除后脉冲伪影。虽然 HPD 的光电探测量子效率低于高性能 SPAD,但所开发的系统可实际用于时域超过几纳秒的双色 ns-FCS。利用新方法观察了蛋白质 A 的 B 结构域在展开状态下的快速链动力学。
Simple and Efficient Detection Scheme of Two-Color Fluorescence Correlation Spectroscopy for Protein Dynamics Investigation from Nanoseconds to Milliseconds
Nanosecond resolved fluorescence correlation spectroscopy (ns-FCS) based on two-color fluorescence detection is a powerful strategy for investigating the fast dynamics of biological macromolecules labeled with donor and acceptor fluorophores. The standard methods of ns-FCS use two single-photon avalanche diodes (SPADs) for the detection of single-color signals (four SPADs for two-color signals) to eliminate the afterpulse artifacts of SPAD at the expense of the efficiency of utilizing photon data in the calculation of correlograms. Herein, we demonstrated that hybrid photodetectors (HPDs) enable the recording of fluorescence photons in ns-FCS based on the minimal system using two HPDs for the detection of two-color signals. However, HPD exhibited afterpulses at a yield with respect to the rate of photodetection (<10–4) much lower than that of SPADs (∼10–2), which could still hamper correlation measurements. We demonstrated that the simple subtraction procedure could eliminate afterpulse artifacts. While the quantum efficiency of photodetection for HPDs is lower than that for high-performance SPADs, the developed system can be practically used for two-color ns-FCS in a time domain longer than a few nanoseconds. The fast chain dynamics of the B domain of protein A in the unfolded state was observed using the new method.
期刊介绍:
ACS Physical Chemistry Au is an open access journal which publishes original fundamental and applied research on all aspects of physical chemistry. The journal publishes new and original experimental computational and theoretical research of interest to physical chemists biophysical chemists chemical physicists physicists material scientists and engineers. An essential criterion for acceptance is that the manuscript provides new physical insight or develops new tools and methods of general interest. Some major topical areas include:Molecules Clusters and Aerosols; Biophysics Biomaterials Liquids and Soft Matter; Energy Materials and Catalysis