[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 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.
Lin
Dan-Ying
DY
Key Laboratory for Atomic and Molecular Nanosciences of Ministry of Education, Department of Physics, Tsinghua University, Beijing 100084, China. dylin@szu.edu.cn
Liu
Xiao-Chen
XC
Wang
Peng-Fei
PF
Ma
Wan-Yun
WY
D001583:Benzoxazoles; D004247:DNA / Q000737:chemistry*; D003952:Diagnostic Imaging; D005456:Fluorescent Dyes; D007834:Lasers; D008856:Microscopy, Fluorescence; D038761:Photobleaching; D011806:Quinolinium Compounds; D059629:Signal-To-Noise Ratio
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA