Fluorescence Imaging Of Single Molecules And Its Applications

Single molecules detection (SMD) is one of the important techniques in the field of analytical chemistry. SMD is one of methods with the highest sensitive detection. It can be used to monitor the reaction pathway and trace the conformations dynamic of large biomolecules. The fluorescence imaging of single molecules is one of ways to detect individual molecules. It was realized and applied in this thesis, which was described as follows.A single molecules fluorescence imaging instrument was built up. It was calibrated by imaging singleλDNA molecules and Rhodamine 6G. Thus, the fluorescence imaging methods for single molecules labeled with multi-dyes and single dye were respectively realized. These two kinds of molecules were independently applied to studies of hydrodynamic and molecules interactions.After the validity of hydrostatic pressure used in microchannel was confirmed, a laser-induced fluorescence imaging method was developed to simultaneously measure flow velocities in the middle and near wall of a channel with particles or single molecules, by selectively switching from the wide field excitation mode to the evanescent wave excitation mode. The measured velocities of particles or single molecules in two positions in a microchannel were consistent with the calculated value based on Poiseuille flow theory. The collisions of microbeads with channel walls were directly observed. It is especially important that the technique can be permitted to measure both velocities in the same experiment to eliminate possible experimental inconsistencies.Series samples of oligonucleotides labeled with single Fluorescin isothiocyanate...