| 光谱学与光谱分析 |
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| Study on Real-Time Imaging of Single Stretched DNA Molecules by Total Internal Reflection Fluorescence Microscopy |
| LIN Dan-ying1, 2, LIU Xiao-chen1, WANG Peng-fei1, MA Wan-yun1* |
1. Key Laboratory for Atomic and Molecular Nanosciences of Ministry of Education, Department of Physics, Tsinghua University, Beijing 100084, China
2. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education/Guang Province, Institute of Optoelectronics, Shenzhen University, Shenzhen 518060, China |
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Abstract 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 real-time 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.
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Received: 2009-04-18
Accepted: 2009-07-22
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Corresponding Authors:
MA Wan-yun
E-mail: mawy@tsinghua.edu.cn
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