Light-sheet Fluorescence Microscopy Based On Scanning Bessel Beam

Light-sheet fluorescent microscopy(LSFM)can acquire information from different depth into the specimen by slice-wise optical sectioning sample with a sheet of thin light excitation from the side of it.Fluorescent signal is collected by a detection objective and is then recorded on a sensor array perpendicular to the light-sheet.Volumetric information is constructed from image stack acquired after the light-sheet scanning through the sample.Compared with confocal laser scan microscopy LSFM bears the virtue of fast optical sectioning capability,low photobleaching and phototoxic,becoming a powerful three dimensional imaging tool in the field of life science.Currently the majority of LSFM systems adapt Gaussian light-sheet as excitation light,thus the expansion to the field-of-view(FOV)has to be at the cost of axial resolution.After all,as considerably as scanning Bessel beam light-sheet expands the FOV,the axial resolution and signal to noise ratio has rarely been improved due to out-of-focus background excited by concentric side lobes of Bessel beam.How to expand the FOV without sacrificing axial resolution is an exigent issue to be addressed.In this dissertation a complementary beam to Bessel beam is proposed and generated,with which a large FOV as well as high axial resolution LSFM system is achieved by sequentially exciting sample with scanning Bessel beam light-sheet and its complementary beam light-sheet,taking the subtraction between the two stacks of images to eliminate out-of-focus background.This dissertation mainly includes following aspects:1.The complementary beam of Bessel beam was constructed and optimized.Aiming at addressing the issue of out-of-focus excitation caused by the side lobes of Bessel light-sheet,we conceived Bessel beam's complementary beam(CB),the scanning of which is exactly the side lobes of Bessel light-sheet.According to intensity distribution characteristics the complementary light-sheet is subjected to,we suggested the general spectrum form of CB after inquiring the spectra of Bessel beams and Mathieu beams.And then we acquired the optimal spectrum expression of CB with genetic algorithm to approximate,within the vicinity of detection objective focal plane,the side lobes of Bessel light-sheet to utmost.A large,uniform and thin light-sheet was obtained after subtraction between two light-sheets.2.The theoretical analysis and optical emulation for an entity of scanning light-sheet fluorescent microscopy were accomplished.We calculated,under the framework of scalar diffraction theory and with chirp z transform,the three-dimension intensity distributions for Gaussian,Bessel and CB light-sheets.We then analyzed point spread functions and their corresponding modulation transfer functions for three different light-sheets.The simulation on imaging process manifests that subtraction method with CB light-sheet employed exhibits high axial resolution comparable to Gaussian light-sheet over the whole FOV of Bessel light-sheet.3.A versatile scanning light-sheet fluorescent microscopy based on spatial light modulation was built up to achieve high axial resolution and large FOV volumetric imaging.We designed and constructed versatile LSFM enabling the scanning of a variety of light fields with the employment of a phase only spatial light modulator into spatial light modulation module,the wave front encoded plane wave out of which is then coupled into scan mirror module.In order to analyze imaging performances among different light-sheet excitations,the Gaussian,Bessel and light needle light-sheets were respectively applied to the imaging of 4?m-diameter fluorescent beads and e-GFP labeled mouse brain section.