Research On Wide-field And High-resolution Fresnel Ptychographic Microscopy

With the rapid development of our country’s manned space project,as one of the most important factors restricting human beings’long-term space exploration and residency,the impact of the space irradiation environment on living bodies has attracted much attention.In the project carried by our school,"Space Environment Ground Simulation Facility",one of the core tasks is to detect the side effects of unlabeled cells after proton microbeam irradiation,thereby revealing the damage laws and mechanisms of space radiation.RIBE refers to the phenomenon that the irradiated cells produce damage signals and induce similar damage to the unirradiated cells in a large area outside the irradiated area.The effective range of the RIBE of biological cells is up to 7.5mm,the detection resolution needs to meet the sub-micron level,and the corresponding information throughput exceeds the limit of the spatial bandwidth of traditional microscopes.In addition,in the in-situ detection scene,traditional illumination methods have introduced serious non-uniform illumination defects;on the other hand,in the offline detection scene,further observation of cell details requires the imaging system to have a higher resolution.Therefore,the core difficulty of the particular microscopic instrument for detecting RIBE is that the microscopic system needs to consider the requirements of a large field of view,high resolution,label-free cell imaging,and resistance to illumination defects.Traditional wide-field microscopy imaging technology cannot solve the problems mentioned above at the same time.This demand is also a new challenge in the field of modern microscopy instrument technology.The Fresnel ptychographic microscopy technology based on speckle illumination has the advantages of a large imaging field,capable of quantifying phase imaging of unlabeled cells,insensitive to illumination defects,and independent of prior information of the illumination light field.It is widely used to image transparent cells.However,the resolution of this technology is still limited by the pixel size of the detector and the collection aperture,which cannot meet the requirements of high spatial bandwidth product imaging for the detection of RIBE.In response to the above problems,the main tasks of this paper are as follows,(1)In order to meet the needs of large-field high-resolution anti-irradiation in-situ detection of the side effects of biological cells,aiming at the problem that the spatial bandwidth product of the existing speckle illumination lensless ptychographic microscopy is limited by the Nyquist sampling theorem,the mechanism of pixel super-resolution Fresnel ptychographic lensless microscopy(PSR-FPLM)has been studied based on the principle of upsampling quantitative phase imaging.This thesis uses random speckle patterns to modulate the complex amplitude of the sample,and scanns the sample laterally to increase the diversity of the diffraction image.Then,this thesis establishes an objective function of pixel super-resolution quantitative phase imaging based on the forward propagation of the light field and the down-sampling process of the detector,and then proposes the pixel super-resolution reconstruction algorithm,constructs the correction coefficient matrix to realize the upsampling correction of the low-resolution sample’s complex amplitude,and finally solves the problem of resolution limited by pixel size while suppressing illumination defects,thereby providing a combination of high spatial bandwidth product and quantitative phase for imaging scenes such as in-situ detection of RIBE.Principle experiments show that the PSR-FPLM technology improves half-pitch resolution from 1.67μm to 0.78μm,the measurement error of the 1.53rad phase sample is 7.84%,and it is highly robust to illumination defects.(2)In order to meet the requirement of a large field of view and high-resolution off-line detection of the side effects of biological cells,aiming at the problem that the spatial bandwidth product of the existing speckle illumination lens-based ptychographic microscopy is limited by the numerical aperture of the collecting objective lens,the mechanism of optical super-resolution Fresnel ptychographic lens-based microscopy(OSR-FPLBM)has been studied based on the principle of random frequency shift of coherent speckle illumination.First of all,the thesis introduces the object defocus to enhance the phase transfer function of the oversampling system,uses a dense speckle pattern to illuminate the complex amplitude of the sample,establishes an objective function of optical super-resolution quantitative phase imaging,and proposes an optical super-resolution algorithm that exceeds the diffraction limit of the collection objective based on the principle of synthetic aperture.Then,in a low-magnification,under-sampling lens imaging system with a larger field of view,the imaging degradation model of the speckle illumination under-sampling lens system is established by combining the low-pass filtering of the light field and the detector down-sampling process,and the optical super-resolution algorithm in under-sampling condition is proposed further to improve the spatial bandwidth product of the imaging system.This method finally provides a microscopic imaging technology with higher resolution,high spatial bandwidth product and quantitative phase imaging for the off-line detection of RIBE.The principle experiment shows that in the under-sampling lens system with2.76×,NA=0.3,OSR-FPLBM achieves a synthetic aperture of 0.6 and improves the half-pitch resolution from 1.25μm to 0.41μm.(3)Based on the method proposed above,comprehensive experiments of high spatial bandwidth product imaging of PSR-FPLM technology and OSR-FPLBM technology were carried out.Unlabeled transparent biological samples such as mouse kidney cells and inner cheek cell samples were tested in the experimental system,which verified that the proposed method can achieve high spatial bandwidth product quantitative phase imaging.Comprehensive experiments show that PSR-FPLM technology achieves a half-pitch resolution of 0.78μm and FOV=6.44mm×4.62mm in a lensless system,and the corresponding spatial bandwidth product is increased from1.07×10~7 pixels to 4.89×10~7 pixels;OSR-FPLBM technology realizes FOV=3.06mm×2.56mm in the lens-based system,the half-pitch resolution is 0.41μm,and the corresponding spatial bandwidth product is increased from 4.60×10~6 pixels to4.66×10~7 pixels.