The Research On Fluorescence Spectrum Imaging And Photobleaching At Single Molecule Level

Single molecule spectrum can be used to distinguish the molecules labeled withdifferent fluorophores in the multicolor components system, to observe and detect thechange of micro-environment. The single molecule spectrum is helpful to obtained themore exact result of fluorescence resonance energy transfer (FRET) and can detect theFRET with single detector. FRET can measure the distance in the 1~10nm range and itfills up the resolution blank of the Near and the Far Field Optical nano-Microscope.FRET has been extensively used to measure distance changes and kinetics and detectthe interaction of the molecules in various biomolecular systems.In the single molecule detection, although hardly any biological moleculeintrinsically fluoresces at convenient excitation wavelengths, a biomolecule can belabeled with fluorophore(s) in a controlled manner for optical investigation. Thestudy of biomolecules has become an important application of single-moleculedetection. However, the time scales and the range of optical experiments on biologicalsystems at room temperature are considerably limited by photobleaching. For example,the photobleaching limits the long-time tracking of the fluorophore in the cell.Bleaching is an irreversible conversion of a fluorescent molecule or particle into anonfluorescent entity. The research on photobleaching can provide the helpful theoryfoundation. For several decades, the researchers contribute to discovery the priciple ofthe photobleaching of the fluorophores. However, until now, most of the principle arenot clear. In the former research of the photobleaching, many work focus on restrainingthe photobleaching, but few researcher studied on the fluctuation of the fluorescenceintensity before photobleaching. In this work, several factors that can effect thephotobleaching of the single Rhodamine B molecule is discussed.Our main works are as follow:1,Study on the single molecule fluorescence spectrum. By placing one transmitgrating before the the chip of the EMCCD in the wild-field fluorescence microscopy,one zero order point and one first order streak can be obtained. Two fluorophors, theAlexa 488 and the Alexa 594, are models to research the spectrum resolution, and theQD525 is used to validate the result of spectrum resolution. The position of the zeroorder point is same as the position without the grating in the optical path. Thedifference between the highest fluorescence intensity of the zero order point and thefirst order streak can be changed into light wavelength by multiply the factor ofspectrum resolution. Then the single molecule spectrum can be obtained and it isconsistent with the coreresponding bulk fluorescence spectra. The single molecule spectra can used to distinguish the multicolor components system, to detect thefluorescence resonance energy transfer (FRET), to research the Localized SurfacePlasmon Resonance and so on.2,Research on the FRET of proteins. The fluorescence resonance energytransfer has been extensively used to research the structure change of the biomolecule,the dynamics and the interaction of molecules. In this work, we use the transmit gratingto detect the FRET with single detector to observe the interaction between the BSAlabeled with biotin and the Streptavidin.3,Research on the photobleaching of Rhodamine B at the single molecule level.We observe the difference in the emamble and single molecule level of Rhodamine Bdissolved in the enthanol, water and four different pH value buffer. Also thephotobleaching of the Rhodamine B is researched to observe the fluorescence intensitybefore quenching, and one conclution was obtained that the change of the fluorescenceintensity has a linear relationship with time, and the distribution of the slope of thesingle step photobleaching accords to Lorenz distribution.