拉直的单个DNA分子的全内反射荧光实时成像研究

全内反射荧光(TIRF)成像技术利用穿透深度仅200 nm左右的隐失波来激发诱导荧光，探测灵敏度和图像信噪比大大提高，成为单分子研究的有力工具。分子梳技术利用DNA末端与固体表面的结合力和周围流体流动产生的侧向力将DNA分子拉伸并平铺在表面上。结合这两种技术，对分子梳拉直的单个DNA分子进行了清晰的实时荧光成像，发现TIRF成像条件下DNA分子与荧光探针YOYO-1组成的复合体可自然避免发生光敏断裂现象;实时监测了单个DNA-YOYO-1复合体的光漂白过程，通过对激发光照射时间与探测器曝光时间进行同步控制，可大幅降低光漂白程度，为拉直的单个DNA分子的长时间实时观察和成像研究优化了实验条件，为实时、可视化地研究其与蛋白质相互作用的动力学过程奠定了基础。

Study on Real-Time Imaging of Single Stretched DNA Molecules by Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy (TIRF) is a powerful tool for single molecule study,since only a thin layer of about 200 nanometers is excited by the evanescent wave,resulting in high sensitivity of detection and high signal-to-noise ratio of images.Molecular combing is a convenient and efficient way to stretch DNA molecules with the help of the binding force between DNA molecule and solid surface,as well as the lateral force introduced by ambient fluid flow.In the present paper,real-time fluorescence imaging of single DNA molecules was carried out with these two techniques.Clear images of single stretched DNA were obtained,while photocleavage of DNA-YOYO-1 complex was found to be naturally avoided under TIRF imaging conditions.Photobleaching of the complexes was investigated in real-time,and was greatly reduced by synchronizing the excitation of light (laser) and the exposure of detector (ICCD).The method optimized the experimental conditions for long-lasting realtime observation and imaging of single stretched DNA molecules,so as to lay a foundation for visually studying the kinetic processes of interactions between DNA and proteins.