Research On Axial Resolution Enhancement Of Optical Sectioning Structured Illumination Microscopy Based On Three-dimensional Structured Illumination

With the development of optical microscopy,the three-dimensional(3D)morphological distribution of sample can be restored by 3D microscopy,which becomes a powerful tool for modern biomedicine.As a branch of 3D microscopy,optical sectioning structured illumination microscopy(OS-SIM)has the advantages of fast imaging speed,weak photobleaching and phototoxicity,and flexible and compatible configuration,and is thus suitable for 3D microscopic imaging of living samples.Although the conventional OS-SIM can remove the out-of-focus background from the in-focus signal,the axial resolution of the final reconstructed 3D image is not improved.Aiming at this drawback,the research in this thesis focuses on the theoretical and experimental studies of OS-SIM to improve the system’s axial resolution.The specific research contents are as follows:1.An OS-SIM method based on 3D structured illumination is proposed,which owns the optical sectioning capability with the enhanced axial resolution by 1.4 times.The out-of-focus background in the imaging results and the low axial resolution restrict the ability of 3D imaging of conventional wide-field optical microscopy.Though OS-SIM can remove the out-of-focus background,it does not improve the axial resolution in the final 3D reconstructed image.In this thesis,a three-beam-interference OS-SIM is proposed,namely TBOS,which uses the 3D structured illumination instead of the conventional dual-beam-interference OS-SIM scheme to excite samples,and then the sectioned image can be obtained by the basic theory of TBOS.The axial resolution of the 3D image obtained by TBOS is enhanced by a factor of 1.4 compared to the DBOS method,resulting in superior 3D imaging capability.2.An OS-SIM imaging system based on 3D structured illumination is designed and built.The 3D structured illumination pattern is generated by three coherent beam interference,with the capability of modulating the samples in both lateral and axial directions.A 3D translation stage and a 2D camera are used to move the sample and acquire the image,respectively.The synchronous control of different hardware devices is implemented using the custom-developed control software based on C++language with high precision and high stability.Based on this imaging system,the performance of TBOS is verified by 3D microscopic imaging experiments on samples of 46nm-diameter fluorescence beads and mouse kidney section.Compared with the DBOS method,TBOS can improve the axial resolution by 1.36 times with the background removal ability,and obtain 3D imaging results with high contrast.3.The theoretical principle of three-dimensional super-resolution structured illumination microscopy(3D-SR-SIM)is analyzed based on the 3D structured illumination OS-SIM method.The possibility of improving the multidimensional resolution is discussed.Preliminary experiments on mouse kidney section have shown that 3D-SR-SIM enables the improvement of both axial and lateral resolutions,and further enhances the 3D imaging specification.