Study On Fast Fluorescence Lifetime Microscopy

With the rapid development of fluorescent labeling technology,fluorescence microscopy is widely used in biological imaging because of the advantages of high specificity and non-invasive.Fluorescence lifetime,as one of the parameters of the fluorescent signal,is closely related to the de-excitation rate of the fluorescent molecule from the excited state to the ground state,and can very sensitively reflect the change of the microenvironment around the fluorescent probe molecule or its interaction with other molecules.Compared with traditional fluorescence intensity based imaging,the image contrast of fluorescence lifetime imaging microscopy(FLIM)is fluorescence lifetime.It is not affected by factors such as excitation light intensity,probe concentration,and photobleaching.Quantitative measurement of many biochemical parameters in the microenvironment,such as oxygen content,ions,metabolic state,quencher distribution,fluorescence resonance energy transfer(FRET)efficiency can be achieved by FLIM,so it is increasingly used in biomedicine,material science,chemistry and other fields.The key to the realization of FLIM technology is the detection of fluorescence lifetime.The most widely used detection method of fluorescence lifetime is timecorrelated single photon counting(TCSPC).This method shows advantages of high sensitivity,high time resolution,and high signal-to-noise.However,in order to obtain an accurate fluorescence decay curve for fitting and calculating the lifetime value,the number of fluorescent photons detected in a single excitation cycle is limited to less than one,and meanwhile enough photons are required to be collected.Thus the imaging speed of TCSPC-FLIM is relatively slow.However,in practical applications,on one hand,researchers hope to acquire FLIM data in a time period as short as possible;on the other hand,the study of many biological problems demand fast imaging speed,such as dynamic imaging of moving organelles,vesicle transport trafficking in live cells,protein-protein interactions,etc.Therefore,it is of great significance to develop fast FLIM imaging technology to meet the needs of the above applications.This thesis focuses on how to improve the imaging speed of TCSPC-FLIM,and the main research work is summarized as follows:1.Based on the TCSPC-FLIM technology using acousto-optic addressable scanning,we optimized the synchronization and data storage method during the system acquisition,developed the corresponding data post-processing and fluorescence lifetime image reconstruction method,and thus realized fast FLIM imaging of ROIs of any number and shape with a standard sample of Convallaria.2.We combined acousto-optic addressable scanning,single particle tracking(SPT)and feedback control,optimized the centroid positioning method,the feedback control model and the acquisition synchronization,and achieved tracking and fast FLIM imaging of single moving particles.3.We applied the Bayes analysis(BA)algorithm to the analysis of fluorescence lifetime in low-photon-count data which were collected in single-particle FLIM tracking,and further improved the imaging speed of the system.The innovation points of the thesis' research work include:1.Proposing and realizing an addressable scanning TCSPC-FLIM imaging method for ROIs of any shape and number in FIFO imaging mode,which improves the FLIM imaging speed to a certain extent;2.Achieving feedback-control-based fast FLIM tracking of single moving particles by optimizing the previous feedback-control program and combining with the BA algorithm for lifetime analysis in low-photon-count case,which is expected to provide a new research method for fast tracking of moving targets in biological samples.