The Research And Application Of Large-filed-of-view And High-resolution Light Sheet Fluorescence Microscope

As an important means of observing the microscopic world,optical microscope has always been an essential tool in modern biomedical research.This technique has evolved from low-resolution two-dimensional observation of stained plant cells into high-specificity,high-resolution and multi-dimensional rapid observation of organs or small animals at cellular resolution.As a novel 3D imaging technology,light sheet fluorescence microscope(LSFM)is widely used in biomedical research,due to its low photobleaching/phototoxicity and rapid 3D imaging capability.However,LSFM still suffers the following problems:contradiction between resolution and field of view(FOV),light absorption and scattering from thick tissue in large samples.This dissertation mainly focused on the problems in 3D high-resolution imaging under large FOV of large samples,mainly including as the following:Firstly,based on conventional Gaussian light sheet microscope,we proposed a sub-voxel light sheet microscope(SLSM).Unlike the Z-axis scanning of conventional LSFM,SLSM applied an oblique scanning method.With proper scanning step size,SLSM can generate 3D images with sub-voxel displacement by oblique scanning.Sub-voxel-resolving(SVR)computation was used to group the original low-resolution images,and iteratively calculated the high-resolution 3D images by maximum likelihood estimation(MLE).We used fluorescent microspheres to measure the point spread function(PSF)of SLSM and improved the lateral and axial resolutions by 3-4 times and 2-3 times,achieving high-resolution under large FOV.Then we used this system to imaging 3D cultured NHBE cells,zebrafish embryos and cleared mouse brain to demonstrate its application in biomedical research.Then,by further combining sub-voxel-resolving and multi-view fusion,we proposed a multi-angle-resolving sub-voxel SPIM(Mars-SPIM).Mars-SPIM can generate multiple 3D image stacks with super-resolving capabilities at multi-angles.The SVR computation was applied to each view and generated multiple high-resolution image stacks.Then the high-resolution images were used as inputs for multiview fusion,and isotropic results were finally obtained.Mars-SPIM can effectively overcome the light absorption/scattering of large samples,and achieve isotropic high-resolution in three dimensions.By using the fluorescent microspheres to measure the PSF of the system,the lateral and axial resolution can be improved 3-5 times and 8-10 times,respectively.Then we used Mars-SPIM to image resin-embedded cleared brain slices,intact mouse brain,and agarose-embedded zebrafish and drosophila embryos to achieve isotropic 3D high-resolution imaging of large samples.Finally,we proposed a propagating-path uniformly-scanned light sheet excitation(PULSE)microscopy based on voice coil motor(VCM).An ultra-thin Gaussian light sheet was generated,and the excitation objective was driven by the VCM to scan the light sheet along the propagation axis.By synchronized the movement of VCM and rolling shutter of camera,the Rayleigh range of light sheet was expander to the scanning range of the VCM.Using the fluorescent microsphere to measure the PSF,the PULSE can generate a stable 1.7μm optical section under the FOV of 4× objective(3.3 mm × 3.3 mm),while the Rayleigh range of conventional 1.7-μm thick light sheet is only 28 μm,nearly a hundred times improvement.Then we used PULSE to image the cleared mouse spinal cord and zebrafish embryos,demonstrating the prospects in biomedical applications.