Research On Frequency-shift Super-resolution Optical Microscopy Using Illumination Manipulation

As an imaging tool,the optical microscope has been widely applied to the field of materials,biology and biomedicine due to its advantages of high resolution,fast imaging speed,minimal invasion,convenient operation and specific-structure imaging.However,because of the limit of the optical diffraction,the lateral and axial resolutions have always been limited to about 200 nm and 500 nm,respectively.Meanwhile,with the development of material science and life science,it is of great necessity to improve the resolution of optical microscopy.Over the past few decades,scientists have been committed to the improvement of the resolution of optical microscopy,and finally opened the door to super esolution after their unremitting efforts.However,there is a long way to go from the proposal of any technology to its perfection,and so are all kinds of super-resolution optical microscopy.Up to now,in order to solve the problems and shortcomings of the super-resolution optical microscopy,further related researches are still in full swing.In this dissertation,a series of researches were carried out to improve the resolution of optical microscopy based on frequency shift realized by illumination modulation.Because of the different characteristics of the coherent and incoherent optical imaging systems,the corresponding theories and methods to break the diffraction limit were proposed respectively:for coherent imaging system,surface wave illumination with large transverse wave vector is proposed;for incoherent imaging system,non-uniform illumination was used.Both theory analysis and experimental verification of these two methods were conducted in detail in this dissertation,and the pass-bands of the systems were expanded with corresponding algorithms.In addition to the realization of two-dimensional super-resolution imaging,the improvement of three-dimensional resolution in incoherent imaging was also studied,and three-dimensional super-resolution imaging was achieved.The main innovations of this dissertation are as follows:(1)In coherent imaging,according to the characteristics of large transverse wave vector of surface waves(evanescent wave and surface plasmon wave),the method of using surface waves for illumination is proposed.In addition,the Fourier ptychography algorithm for synthesis and fusion of the frequency bands obtained in different orientations is adopted to expand the pass-band of the system.As a result,the intensity and phase distributions of the object with a resolution of ?/3 are ultimately obtained without the need of a reference beam.In addition,the enhancement of image contrast is realized by using the characteristic of the field enhancement of surface plasmon waves.(2)Aiming at the problem of laser speckle in coherent imaging,a multi-angle ring-scanning illumination method is proposed to eliminate the influence of laser speckle on the imaging quality,and images with high signal-to-noise ratio are acquired.(3)In incoherent imaging,a new patterned-illumination Fourier ptychographic microscopy is proposed.By updating the optical transfer function,the aberration of the system can be corrected digitally.It also has good robustness to noise.With this technique,a time resolution up to 0.5 fps can be achieved,which can realize living cell imaging of the biological samples.(4)In coherent imaging,a set of I5S three-dimensional structured-illumination super-resolution microscope based on scanning galvanometer is designed,which improves imaging speed compared with the traditional grating-type structured-illumination system and the light utilization efficiency compared with the spatial light modulator structured-illumination system at the same time.Moreover,the mechanism of fluorescence saturation effect is studied and applied to the three-dimensional imaging in this dissertation,which offers a three-dimensional resolution of about ?/9.