Fluorescence Lifetime Imaging Based On Light Sheet Microscopy

As an effective method to obtain biological information from the sample,fluorescence microscopy has been an important tool in life science and biomedical research.However,the structural information that can be obtained by ordinary fluorescence intensity imaging is no longer sufficient to researchers already.To understand the mechanisms of life activities,disease development,and drug effects,etc.It often needs to acquire the physiological characteristics including functional information in biological samples.Light sheet fluorescence microscopy(LSFM)and optical projection tomography(OPT)are two fundamental techniques in mesoscopic imaging field.However,to the best of our knowledge,there is no study,to numerically discuss the differences between their image formation properties and to adequately quantify their own strengths and limitations.In this study,we present a numerical analysis and experimental characterization of the spatial resolution to describe the different behavior of the “standard” OPT and LSFM,illustrating the higher lateral but the lower axial resolution of LSFM as compared to OPT.A number of biomedical samples with different size have also been imaged to demonstrate the application performance of both techniques at different spatial scales.For fluorescence lifetime microscopy,time gating is an important method and it has a faster imaging speed and flexibility compared to the others.For fluorescence intensity imaging,LSFM has smaller photo-bleaching and higher image quality compared to other imaging methods in the mesoscopic field.Therefore,to build a LSFM-FLIM imaging system which combines both advantages of light sheet fluorescence microscopy and time gating FLIM have a great significance for the application of multi-dimensional fluorescence imaging.In this thesis,we built a fluorescence lifetime imaging system based on light sheet microscopy technology and presents its applications in biomedical and biotechnological field.The specific contents include the study of the light sheet microscopy,as well as the setup of a time gating fluorescence lifetime imaging system,programmed software control program platform,characterized the imaging system,and researched the application of LSFM-FLIM in biomedical science.The experimental test and imaging application proved the reliability and availability of the imaging system.