Research On SpFRET And Single Fluorescence Molecule Photobleaching

Single molecule detection (SMD) has been applied to study the change of the molecular conformation, dynamics, interactions between molecules and the control of single molecules. SMD can obtain individual information under non-equilibrium conditions and the fluctuation of the system under equilibrium conditions. It also can be use to track the process of microcosmic dynamics which is enshrouded. Besides, the molecules do not have to be synchronized during SMD, and some rare intermediates or transitional states can be catched easily. Using single-molecule fluorescence spectroscopy can distinguish between complex biological systems that labeled with different fluorophores of biological macromolecules.In this thesis, we use techniques of single-molecule fluorescence spectroscopy to detect single-pair fluorescence resonance energy transfer by adding a transmit before the EMCCD. So one zero order point and one first order streak can be obtained. We use a Double-pass filter to make sure emission of both dornor and acceptor can passed throuh ,Finally we achived to detect single pair FRET.Photobleaching is used to be a big obstacle of the widely use of fluorescent dyes. And how to suppress photobleaching is becoming the focus of research, but there are few studys focus on the change of fluorescence intensity before its photobleaching. The bleaching of single dye molecule is considered as single step photobleaching, and the intensity of the fluorescence of the dye before photobleaching is steady. In this work, the photobleaching of single fluorescence dyes are studied. We find that the intensity before photobleaching is not always steady, enhancing and quenching are also found in our research, and most of them are tending to quenching. Dissolved in the different pH buffers and different polar solvents, the distribution of the slope of the single step photobleaching accords to Lorenze distribution.The coat and the DNA of virus are dual-labled. We use Alexa594 and QD525 to lable with the coat of virus. After the virus release DNA, we quickly added YOYO-1 to lable with it, and then oberserved under the microscope.