Super-resolution Imaging And Reconstruction Methods Based On Point Illumination And Freauency Shifting

In the endless journey of exploring the macrocosm,fluorescent microscopes have been widely used in the research of biology,medicine and materials science due to their advantages of low invasiveness,specific identification and deep penetration depth that electron microscope does not have."It is necessary to have effective tools to do good work." So breaking the barrier of optical diffraction limit to achieve higher resolution in studying the internal structure of finer objects structure in detail,exploring the internal mechanism and interaction to promote the development of scientific and technological in related fields and even the entire world is really important for scientific researchers.In the past two decades,although a variety of super-resolution microscopy have emerged,including Stimulated Emission Depletion microscopy(STED),Structured Illumination Microscopy(SIM)and Single-molecule Localization Microscopy(SMLM),they still have their own limitations and problems.This dissertation focuses on the frequency domain.Based on the traditional confocal microscope,I designed and set up a super-resolution system based on point-illumination and frequency shifting,which can obtain high-frequency information through the nonlinear effect of fluorescence excitation,phase modulation and parallel detection.Then I used the frequency shifting algorithm to move the information,which beyond the cut-off space frequency of the collected objective lens,to the detectable area and demodulating the high-frequency information to achieve super-resolution imaging.I also proposes a super-resolution imaging and reconstruction method based on point illumination and frequency shifting,which called Image Scanning Difference Microscopy(ISDM).This method uses a detector array to record the images of sample under solid and hollow illumination pattern.Then the pixel reassignment algorithm is used to obtain the parallel detection results of the two illumination patterns,respectively.Finally,the super-resolution imaging result can be obtained by subtracting the two images.ISDM can not only reduce the artifacts generated by negative values in the subtracting process,but also greatly enhance the resolution.This method realized a transverse resolution of～λ/6(triple diffraction limit)under single wavelength,single incident path and low illumination intensity,which has never been achieved in any other far-field super-resolution microscopy.Another frequency shifting reconstruction method proposed in this dissertation is Saturated Image Fusion Microscopy(SIFM),which combines Nonlinear Focal Modulation Microscopy(NFOMM)with interlaced reconstruction algorithm and parallel detection technology to improve the temporal and spatial resolution,respectively.We achieved a resolution 6 times that of diffraction limit with the same imaging speed as the traditional confocal microscopy.The super-resolution imaging system and reconstruction methods based on point illumination and frequency shifting proposed in this dissertation have the advantages of simple setup,widely sample and low photobleaching and phototoxicity while achieving resolution comparable to STED.It is also easy to upgrade from traditional confocal systems,so I anticipate this system will be a useful observation tool in the biology,medicine,materials science and other research fields.