The Constructing And Functioning Of A System For Fluorescence Lifetime Imaging And Dynamical Analysisat Single Molecule Level

With the development of science and technology, the scientific research is focusing on the information acquisition in dynamics and at single molecule level. Single Molecule Detection(SMD) technology can thus be used to investigate the individual molecule characteristics, distribution and process of movement.As one of the widely used single molecule technique, Single molecule fluorescence imaging and analysis is on the basis of monitoring the fluorescence trajectories and imaging with nanometer resolution to uncover the involved scientific mechanisms. However, this intensity based imaging is easy to be influenced by the factors such as the excitation intensity, the concentration of fluoresce in, light bleaching and so on. The recently developed single molecule fluorescence lifetime imaging(FLIM) can significantly reduce the influence of these factors, and provides more detailed information about the chromophores micro environment and the individual characteristics of single molecule, demonstrating the obvious advantages relative to the fluorescence intensity imaging technology.Fluorescence lifetime as an important parameter of fluorescent signal has been widely used to investigate different fluorescent groups, the energy level structure of the fluorescent groups, and the change of fluorophore micro environment, the binding mode of molecular, intermolecular energy transfer and so on.Fluorescence Lifetime Imaging Microscopy(FLIM) consists of several parts, including pulsed excitation laser with picoseconds resolution, time correlation of single photon counting(TCSPC), confocal fluorescence microscopy, nanometer resolved translation stage, and so on, in order to obtain single molecular dynamics and imaging with high temporal and spatial resolution.This thesis mainly introduces the successful constructing and functioning of FLIM system forfluorescence lifetime imaging and dynamical analysisat single molecule level. The setup system realizes the functions of single molecular imaging with picosecond temporal resolution and nanoscale spatial resolution,initial accesses single molecule fluorescence lifetime imaging of biological macromolecules, the fluorescence dye molecules, luminescent nanoparticles and so on. The thesis is mainly divided into the following several parts:The first chapter summarizes the development and application of single molecule detection and introduces the FLIM technology.The second chapter introduces the main technology. and method used in this thesis, time correlation of single photon technology and confocal laser scanning microscopy, and related techniques.The third chapter describes the construction and optimization of single molecule FLIM and dynamic analysis system in detail. It is proved that this system can be functioned with fluorescence lifetime imagine at single molecule level, the dynamical data acquisition and analysis, single molecule fluorescence and lifetime correlation spectroscopy, single molecule FRET, etc. The temporal resolution of this systemis determined to be less than 50 picoseconds, and spatial resolution is around 200 nm. The part also presents in details about the calibration of optical components, instrumental debugging, function optimizing.The fourth chapter illustrates the application and function extension. The optimized system can realize single molecule fluorescence lifetime imaging(FLIM) and analyze fluorescence resonance energy transfer(FRET), fluorescence correlation spectroscopy(FCS, FLCS), fluorescence cross correlation spectroscopy(FCCS, FLCCS), anti-bunching effect(Anti-bunching) and so on.Briefly, the thesis introduces the constructing and functioning of a system for fluorescence lifetime imaging and dynamical analysis at single molecule level, by combining with time-correlated single-photon counting and confocal fluorescence imaging techniques. The calibration, optimization, debugging, functionextension, and application about this established system are described in detail as well.