Study On Large Field Of View Light-sheet Fluorescence Microscopy Based On Novel Nondiffractive Beams

Light-sheet fluorescence microscopy(LSFM)uses a thin light-sheet to illuminate the sample from the side to excite fluorescence,and then collects the fluorescence through a detection objective placed perpendicular to the light-sheet.By moving the sample axially,or synchronously moving the light-sheet/detection objective,a series of two-dimensional fluorescence images of the sample at different axial positions can be captured,allowing three-dimensional(3D)microscopic imaging.LSFM selectively illuminates the imaging area of the sample,which avoids the generation of defocused background and enables natural three-dimensional tomography.Additionally,it minimizes photodamage and is thus suitable for long-term,three-dimensional microscopic imaging of living samples.Compared to point scanning microscopic imaging,LSFM uses camera to achieve area array detection in wide-field,resulting in fast imaging speed.It has been successfully applied in various fields including developmental biology,functional imaging,cell biology,plant and algae research,whole-brain imaging.There are problems in LSFM such as the mutual constraints of high axial resolution and large field of view,and how to improve the 3D imaging speed.In this thesis,a novel non-diffraction beam is designed and generated.By scanning it to generate light-sheet for imaging,a large field of view is obtained while ensuring high spatial resolution.In addition,a deformation mirror is used in the detection optical path for optical field modulation to extend the depth of field,and the axially scanned light-sheet achieves fast 3D imaging without mechanical movement.The following work and innovations are presented:1.Design and generate a novel non-diffraction beam multiple Airy beams(MAB).Non-diffracting beams,such as Airy beams and Bessel beams,exhibit propagation invariance and self-healing properties.Scanning light-sheet generated by non-diffracting beams enables deeper penetration depth and wider field of view compared to those generated by Gaussian beams.However,the side lobes of non-diffracting beams can cause defocusing excitation and affect imaging quality.To address this issue,MAB are designed and generated by modifying the phase spectrum of Airy beams.Compared to Airy beams,MAB has fewer side lobes and the energy is more concentrated on the focal plane,resulting in improved imaging contrast.MAB retains the non-diffracting properties and can cover the field of view as large as Airy beams.The combination of MAB and complementary beam subtraction(CBS)method can further improve axial resolution.2.A light-sheet fluorescence microscopy system with spatial light modulation function has been designed and built.The system has a tight spatial light modulation module at the illumination side,which allows flexible dynamic generation of different types of light by loading the designed Computer-generated-holograms.In addition,we have designed and 3D-printed a sample chamber,the long working distance excitation objective is mounted outside the chamber,larger chamber space facilitates mounting and movement of standard slides samples.Based on this LSFM system,the performance of MAB is verified by three-dimensional microscopic imaging experiments on samples of fluorescent beads,aspergillus conidiophores and lily pollen,with a field of view: 450μm × 450μm × 200μm,lateral resolution 0.6μm and axial resolution 1.2μm.Compared with Gaussian light-sheet,the field of view is expanded by twelve-times without sacrificing axial resolution.3.A study of rapid three-dimensional microscopic imaging with simultaneous light modulation of the illumination and detection paths has been carried out.The three-dimensional imaging of the axial mechanically moving sample is limited by the speed of the motorized translation stage,with an imaging speed of only 4 frames/s.To increase the speed of three-dimensional imaging,a deformable mirror is used to modulate the light field in the detection path to expand the depth of field,and the light-sheet is scanned axially and captured synchronously with the camera to realize three-dimensional imaging without mechanical movement,which increases the three-dimensional imaging speed by more than ten-times.The Richardson-Lucy method is used for image deconvolution to ensure the quality of microscopic imaging.