The combination of magnetic trap(MT) and fluorescence resonant energy transfer(FRET) allows for nanoscale measurements of configurational changes of biomolecules under force. However, the magnetic bead involved in MT ...The combination of magnetic trap(MT) and fluorescence resonant energy transfer(FRET) allows for nanoscale measurements of configurational changes of biomolecules under force. However, the magnetic bead involved in MT experiments introduces a substantial amount of background fluorescence which reduces the signal-to-noise ratio(SNR) of FRET significantly. Moreover, the short lifetime of the dye used in FRET limits the total sampling time when combined with MT. Here we use a moveable tube lens to adjust the wave front in the light pathway of MT so that both images of the magnetic bead and the fluorescent signals can be detected when long DNA handles are used to reduce the auto-fluorescence of the magnetic bead. We utilize the internal trigger of an electron multiplying charge-coupled device camera to control a shutter so that the dye can be excited intermittently when long time measurement of FRET is needed. As a demonstration of the hybrid technique, we observe the unfolding/refolding dynamics of a DNA hairpin and measure the DNA unwinding activity of the saccharomyces cerevisiae Pif1(Pif1). Our results show that the unwinding burst of Pif1 under external force is different from that without the force. In addition, the improvement provides a better SNR and a longer sampling time in experiments in the MT-FRET assay.展开更多
基金the National Natural Science Foundation of China under Grant No 11574382.
文摘The combination of magnetic trap(MT) and fluorescence resonant energy transfer(FRET) allows for nanoscale measurements of configurational changes of biomolecules under force. However, the magnetic bead involved in MT experiments introduces a substantial amount of background fluorescence which reduces the signal-to-noise ratio(SNR) of FRET significantly. Moreover, the short lifetime of the dye used in FRET limits the total sampling time when combined with MT. Here we use a moveable tube lens to adjust the wave front in the light pathway of MT so that both images of the magnetic bead and the fluorescent signals can be detected when long DNA handles are used to reduce the auto-fluorescence of the magnetic bead. We utilize the internal trigger of an electron multiplying charge-coupled device camera to control a shutter so that the dye can be excited intermittently when long time measurement of FRET is needed. As a demonstration of the hybrid technique, we observe the unfolding/refolding dynamics of a DNA hairpin and measure the DNA unwinding activity of the saccharomyces cerevisiae Pif1(Pif1). Our results show that the unwinding burst of Pif1 under external force is different from that without the force. In addition, the improvement provides a better SNR and a longer sampling time in experiments in the MT-FRET assay.
文摘论述了基于一种1 024×1 024像素EMCCD图像传感器CCD201的高性能分离式远程成像系统设计方法。采用光纤通信技术,系统分为成像端与控制端,使系统可用于远程监测场合。利用CPLD器件产生CCD逻辑时序及视频同步处理控制时序,采用集成器件与分立器件相结合的方式实现EMCCD垂直驱动时序,获得视频信号;使用带有CDS功能的16位模数转换器对CCD视频信号进行数字化;数字化图像经由光纤发送到远程控制采集端。采用全双工通信,实现电子增益、放大倍数、温度等参数的远程控制。文中详细介绍了驱动电路、视频处理电路、数据远程交互单元与控制采集单元等关键组成部分的技术实现方法。设计的成像系统采用USB方式进行数据实时采集,能够连续获取16位数字图像数据,光动态范围大于70 d B,增益倍数最高能够大于0.1e-/ADU,控温精度达到±0.5℃。设计方法可应用于类似CCD成像系统的设计中,并使系统能够在微光成像及辐射场诊断中应用。