Construction And Experiment Of Dual-color Confocal Fluorescence Lifetime Measurement System

Fluorescence lifetime is sensitive to the microenvironment of the fluorophore,and is not affected by excitation light intensity,fluorophore concentration and photobleaching.It can provide biological functional information complementary to fluorescence spectroscopy and fluorescence microscopy.Fluorescence lifetime measurement system is usually established based on laser scanning confocal microscopy(LSCM).With sub-micron resolution,LSCM has been widely used in the fields of life science,biomedicine,industrial detection,and so on.Confocal pinholes in the LSCM can remove all emission not originating from the focal plane,thus providing three-dimensional optical imaging capability.Two-photon laser scanning microscope(TPLSM)combines LSCM and two-photon excitation technology.The two-photon excitation is based on the third-order nonlinear effect and only occurs at the focal point of the objective lens,therefore three-dimensional images can be obtained without confocal pinholes,which improves the fluorescence detection efficiency.In addition,TPLSM has the advantages of non-invasivity,deep imaging depth,high imaging quality,and is suitable for observation of living cells,so it is an important tool for in vivo imaging of biological tissues.In this paper,a fluorescence microscopy system with dual-color LSCM imaging mode and TPLSM imaging mode was built.Then,the fluorescence lifetime measurement experiment was carried out in the dual-color LSCM imaging mode to obtain the fluorescence intensity decay curves of e GFP and m Cherry.Finally,the fluorescence lifetimes of e GFP and m Cherry were analyzed using three fluorescence lifetime fitting methods of L-M algorithm,FFT algorithm and Prony algorithm.The main contents are as follows:(1)Construction of dual-color LSCM/TPLSM system.The fluorescent microscopic imaging system mainly includes: LSCM561 nm excitation light path,LSCM470 nm excitation light path,LSCM detection and lifetime measurement light path,and TPLSM excitation and imaging light path,among which the first three light paths constitute the dual-color LSCM imaging system.In the two excitation paths of LSCM,the issues including the overlapping of the dual-color excitation spots and the low coupling efficiency of the optical fiber were mainly solved.Two detection modes were designed in the LSCM detection and lifetime measurement light path,one for fluorescence intensity detection,and the other for fluorescence lifetime detection with dual channels that can be used to simultaneously observe two different fluorophores in the sample.In the TPLSM excitation and imaging optical path,the femtosecond laser was splitted into two beams and one of them was introduced into the beam control unit and finally into the MPM200-2two-channel multi-photon system to realize the excitation and fluorescence detection of the sample.(2)Experiment of fluorescence lifetime measurement.The experiment was carried out under the dual-color LSCM imaging mode.The ligand DKK1(labeled with e GFP)of different concentrations were bound to the cell membrane receptor LRP6(labeled with m Cherry),respectively.The fluorescence decay curves of e GFP and m Cherry were obtained based on dual-channel time-correlated single photon counting(TCSPC).Then the L-M algorithm,FFT algorithm and Prony algorithm were used to fit the fluorescence lifetime,and calculate the lifetime values of e GFP and m Cherry.The results showed that the fluorescence lifetimes of e GFP and m Cherry obtained by the three algorithms were consistent,and agreed well with the literature reports.The fluorescence lifetime of e GFP fluctuated around 1.9 ns at different ligand concentrations,while the fluorescence lifetime of m Cherry was between 1.5 ns and 1.6 ns.In addition,the mean fluorescence lifetime of e GFP decreased with the increase of ligand concentration,while the mean fluorescence lifetime of m Cherry increased slightly with the increase of ligand concentration.When the ligand DKK1(e GFP labeled)of different concentrations were bound to the cell membrane receptor LRP6(m Cherry labeled),the number of LRP6 protein reduced,and the microenvironment of e GFP and m Cherry changed,resulting in the change of their fluorescence lifetime.