Study On Fluorescence Lifetime Tracking Of Moving Particles

Fluorescence microscopy can be used to obtain structural and functional information of fluorescence labelled substances by detecting the intensity,polarization,spectrum and lifetime of the fluorescence signal.Because fluorescence lifetime is sensitive to any interaction or energy transfer process between an excited molecule and its environment,and is usually independent of probe concentration,excitation power and photobleaching,fluorescence lifetime imaging microscopy(FLIM)is often used to measure many biophysical and biochemical parameters of the microenvironment of fluorescent probes,such as concentrations of ions or oxygen,pH value,hydrophobicity of solution,distribution of quenchers,etc.Therefore,FLIM has been widely applied in biology and microbiology.Time-correlated single photon counting(TCSPC)is the most commonly used technique for fluorescence lifetime measurement in FLIM.TCSPC-FLIM uses a pulsed laser to excite samples,detects and records the arrival time of each single fluorescence photon at the detector after the excitation pulse,accumulates the photon arrival times to achieve a histogram of the number of fluorescence photons with time for each pixel and fits it with the exponential decay curve to obtain the lifetime and its distribution.TCSPC-FLIM has such advantages as high signal to noise ratio,high time resolution,high sensitivity and wide dynamic range,and becomes the most widely used FLIM technique.However,a conventional TCSPC-FLIM system usually implements a twodimensional galvanometer as its scanning module.Due to the inevitable mechanical inertia of the galvanometer and the inflexible scanning strategy,the imaging speed of TCSPC-FLIM is limited to some extent so that it is not good enough to carry out realtime recording of some rapid changes in living cells,and is impossible to track and measure lifetime of moving particles in the cell.In fact,when the object is a single moving particle,the scanning area of the laser should be restricted to be really small and it should be moved with the particle in order to realize the tracking and lifetime detection of the particle,which means a more flexible and stable scanning method should be used.Therefore,we employed a system based on a two-dimensional acoustooptic deflector(AOD)instead of a galvanometer as the scanning module.The AODFLIM system was used to achieve fluorescence lifetime tracking of moving particles.To realize a rapid tracking and dynamic fluorescence lifetime detection of a single moving particle,we introduced a wide-field detection,a particle tracking algorithm and a feedback control to the AOD-FLIM system.Based on Lab VIEW development language,we wrote the wide-filed detection controlling code,the centroid localization algorithm,the feedback control code and the AOD-based addressable scanning code and integrated them with the TCSPC acquisition and data analysis software,and developed a system for moving particle tracking and dynamic fluorescence lifetime detection.With this system,we can simultaneously track the motion trajectory and record the dynamic lifetime information for a single moving particle,which may provide a new tool for cell biology studies such as the study of intracellular biological macromolecules and their interactions with the surrounding environment or structure.The main works of this thesis are listed as follows:1.An addressable scanning two-photon excitation AOD-FLIM system has been set up,which has the ability to image any region of interests on the sample.Results of single-channel and three channel synchronization were compared and analyzed to optimize the synchronization mode for moving particle fluorescence lifetime tracking.Calibration experiments using fluorescent beads were carried out to verify the reliability of the system and the feasibility of reflecting the variation of the microenvironment along the motion trajectory by collecting fluorescence lifetimes of moving particles.2.We proposed and realized a method to simultaneously acquire the trajectory and dynamic fluorescence lifetime of moving single particles by introducing a wide-field detection,a particle tracking algorithm and a feedback control to the previous AODFLIM system.We wrote and integrated the wide-filed detection controlling code,the centroid localization algorithm,the feedback control code and the AOD-based addressable scanning code into a system control software to realize the track scanning of the moving particle and obtain the motion trajectory and dynamic lifetime information simultaneously.3.Fluorescence beads with Brownian movement in pure glycerol were used to calibrate the system to demonstrate its capability for single particle tracking and dynamic fluorescence lifetime acquisition.By reconstructing the tracking and detecting results,we simultaneously acquired the trajectory and lifetime of the moving particle.Analysis of the results showed that the system is capable of tracking and detecting particles' Brownian movement.The innovation of this thesis is summarized as follows:1.We proposed and realized a method for tracking moving particles and obtaining dynamic fluorescence lifetime information by combining the AOD addressable scanning,TSCPC technique,wide-field imaging,single particle tracking algorithm and feedback control.2.We proposed a microenvironment measurement method based on fluorescence lifetime tracking of moving particles,which may provide a new tool for the study of cell biology.