Sano Yutaka, Itoh Yuji, Kamonprasertsuk Supawich, Suzuki Leo, Fukasawa Atsuhito, Oikawa Hiroyuki, Takahashi Satoshi
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan.
Department of Chemistry, Graduate School of Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
ACS Phys Chem Au. 2023 Nov 10;4(1):85-93. doi: 10.1021/acsphyschemau.3c00040. eCollection 2024 Jan 24.
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) much lower than that of SPADs (∼10), 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.
基于双色荧光检测的纳秒分辨荧光相关光谱技术（ns-FCS）是研究用供体和受体荧光团标记的生物大分子快速动力学的有力策略。ns-FCS的标准方法使用两个单光子雪崩二极管（SPAD）来检测单色信号（四个SPAD用于检测双色信号），以消除SPAD的后脉冲伪像，但代价是在计算相关图时利用光子数据的效率。在此，我们证明了混合光电探测器（HPD）能够在基于使用两个HPD检测双色信号的最小系统中记录ns-FCS中的荧光光子。然而，HPD产生后脉冲的产率相对于光检测速率（<10）远低于SPAD（~10），这仍可能妨碍相关测量。我们证明了简单的减法程序可以消除后脉冲伪像。虽然HPD的光检测量子效率低于高性能SPAD，但所开发的系统可实际用于几纳秒以上时域的双色ns-FCS。使用新方法观察到了未折叠状态下蛋白质A的B结构域的快速链动力学。
