Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors
Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors
复制标题
基于线共焦光学系统与混合光电探测器相结合的微秒分辨率单分子 FRET 时间序列测量
DOI：
10.1039/c7cp06268k
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发表时间：
2018
影响因子：
3.3
通讯作者：
Satoshi Takahashi
中科院分区：
文献类型：
--
作者：
Hiroyuki Oikawa;Takumi Takahashi;Supawich Kamonprasertsukac;Satoshi Takahashi
基于线性共焦光学系统的单分子荧光时间序列测量是研究生物大分子结构、动力学和异质性的有力手段。这种方法能够从单个荧光团中每毫秒检测数千个荧光光子，这意味着测量荧光共振能量转移（FRET）效率的潜在时间分辨率为10 μs。然而，在该方法中使用成像光电探测器的必要性将FRET效率测量的时间分辨率限制为约100 μs。在这项研究中，一个新的光电探测器称为混合光电探测器（HPD）被纳入线共焦系统，以提高时间分辨率，而不牺牲的时间序列检测的长度。在所考察的几种设置中，基于10 μm狭缝宽度和高速计数装置的系统充分利用了线共焦光学系统和HPD的特点。该方法在sm-FRET时间序列测量中实现了10 μs的时间分辨率和约5 ms的观察时间。所开发的装置用于蛋白A的B结构域的天然状态。
Single-molecule (sm) fluorescence time series measurements based on the line confocal optical system are a powerful strategy for the investigation of the structure, dynamics, and heterogeneity of biological macromolecules. This method enables the detection of more than several thousands of fluorescence photons per millisecond from single fluorophores, implying that the potential time resolution for measurements of the fluorescence resonance energy transfer (FRET) efficiency is 10 μs. However, the necessity of using imaging photodetectors in the method limits the time resolution in the FRET efficiency measurements to approximately 100 μs. In this investigation, a new photodetector called a hybrid photodetector (HPD) was incorporated into the line confocal system to improve the time resolution without sacrificing the length of the time series detection. Among several settings examined, the system based on a slit width of 10 μm and a high-speed counting device made the best of the features of the line confocal optical system and the HPD. This method achieved a time resolution of 10 μs and an observation time of approximately 5 ms in the sm-FRET time series measurements. The developed device was used for the native state of the B domain of protein A.