Point Spread Function Engineering And Its Application To High/super-resolution Microscopy Imaging

The development of life sciences and biomedicine has placed increasing demands on optical microscopy imaging,however,the imaging performance of optical systems has been limited by the Abbe diffraction limit.Although researchers have developed various super-resolution microscopic imaging techniques in recent decades to suit the needs of different application scenarios,there are still many shortcomings in the existing optical microscopic imaging techniques.In high/super-resolution optical microscopy,the point spread function reflects the resolution of optical microscopy.Modulating the point spread function provides a more direct and effective technical method to achieve high/super-resolution microscopic imaging.This thesis studies the use of point spread function modulation to improve the three-dimensional resolution of optical microscopy systems and eventually break the optical far-field diffraction limit and proposes some new methods and theories for optical high/super-resolution microscopic imaging techniques.The full paper achieves high/super-resolution microscopic imaging and large axial single-molecule localization by modulating the point spread function in the spatial and Fourier domains.The main research of this paper includes:1、The computational models of illumination point spread function and detection point spread function of the point-scan imaging system are established based on the vector diffraction integral,which provides a theoretical basis and analytical tools to study the performance improvement of optical microscopy imaging.In this paper,a point spread function differential coherence high-resolution imaging technique is proposed to improve the axial resolution of point scanning confocal imaging by 5 times and the lateral resolution by 1.7 times.The relevant experimental system is built,and the results of simulation calculations are verified by experiments on nano-microspheres and cellular microtubules.2、To overcome the complexity of structured illumination systems and the possible phase errors of sequential illumination patterns required for super-resolution image reconstruction,we propose Fourier Ptychographic reconstruction for point-scanning virtual structure detection microscopy(Theta-FP-vPSM).Using the virtual structure detection technique,the original data acquired by the optical system is multiplied with the digital modulation function to obtain the structured stripe modulated image,and the Fourier Ptychographic algorithm(Theta-FP)is used to obtain the high-frequency information to reconstruct the super-resolution image.The digital modulation function can be designed on demand for more flexibility and accuracy.In addition,the Theta-FP-vPSM imaging technology combines the performance of conventional SIM super-resolution with the performance of confocal layer sectioning,solving the problem that SIM microscopy systems cannot image thick samples.3、To address the problem of slow imaging speeds of point-scanning virtual structure detection microscopy,the Fourier Ptychographic reconstruction-based line-scanning structure detection super-resolution microscopy(PHI-FP-vLSM)is proposed to substantially improve the imaging speed.For the problem that the Theta-FP reconstruction algorithm will lose some information and make the final reconstructed image incomplete,the PHI-FP reconstruction algorithm based on the cosine function modulation with different spatial frequency and phase changes are proposed for the reconstruction of vLSM super-resolution images.4、Since the Fourier Ptychographic algorithm requires several iterations with constraints imposed between the spatial domain and the Fourier domain,it leads to a slow reconstruction speed of super-resolution images.To shorten the time of reconstructing super-resolution images,a nonlinear reconstruction algorithm is proposed,which only needs to calculate in the spatial domain to recover high-frequency information,greatly improving the speed of super-resolution image reconstruction,and providing a basis to assist Theta-FP and PHI-FP to achieve real-time observation.5、A combined vortex phase is proposed to generate a double-helix PSF with an axial positioning depth of about 10μm at high numerical apertures,and a corresponding optimization algorithm is developed based on the Fresnel approximation imaging principle.The optimized combined vortex phase is used to localize the fluorescent molecules,and the high-precision 3D localization imaging with an axial detection depth of 10 μm can be achieved at a numerical aperture of 1.4,which provides a basis for 3D single-molecule localization super-resolution imaging.