Research Of Wide-field Super-resolution Optical Fluctuation Imaging Technology

It's tough to break through the limitation of the resolution in wide-field fluorescence microscopy(WFM),by optical diffraction typically to 200 nm laterally,although a number of techniques have been recently developed,which still face many shortcomings.Compared with stimulated emission depletion(STED),photo-activated localization microscopy(PALM),stochastic optical reconstruction microscopy(STORM)and saturated structured illumination microscopy(SSIM),the super-resolution optical fluctuation imaging(SOFI)does not require specialized equipment and high excitation intensity to achieve robust three-dimensional resolution enhancement unlimited by optical diffraction.However,the effect from difference of blinking processes and photonics shot noise,limitation by pixel size of the CCD camera and difficulty in computing high-order cumulants make high-order SOFI hard to implement.Its application is quite limited when the requirements can't be met in low-order SOFI.Aiming at problem of photonics shot noise and computational difficulty in high order SOFI,this paper proposes a zero-time-lag auto-cumulant method based on low-pass denoising,which is shot noise suppressive and more adaptive for high order SOFI.Furthermore,the suppression of random noise and limitation of resolution by pixel size is studied in this paper.Finally,experimentally measure the three-dimensional resolution of SOFI.The main works are as follows:1.Studying the super-resolution optical fluctuation imaging(SOFI)and balanced super-resolution optical fluctuation imaging(b SOFI).A theoretical model of SOFI is established to analyze the resolution enhancement and effect of blinking difference in the image.Besides,the theoretical model of b SOFI is established,and the numerical simulation of its data processing is carried out to analyze the improvement of SOFI.2.Research on suppression of noise in SOFI.Analyzing the damages to SOFI image of random noises generated in WFM,and discussing the suppression of photonics shot noise through auto-cumulant with time lag and spatialtemporal cross-cumulant.Then,a zero-time-lag auto-cumulant method based on low-pass denoising,which is more suitable for high order SOFI is proposed,and the suppressing effects on photonics shot noise of different algorithms are compared through simulation.3.Methods to surpass the limitation of the pixel size of CCD camera.Models of interpolating virtual pixels through spatialtemporal cross-cumulant and Fourier interpolation,and the improvements of spatial sampling frequency are demonstrated by simulation.4.Verification experiments of SOFI.The 2nd-order and 4th-order SOFI experiments show 1.41-and 1.98-fold improvements in lateral resolution respectively,which are consistent with the theoretical value.Brightness and blinking response are linearized,and a 1.78-fold lateral resolution improving linearly with the cumulant order is obtained in 2nd-order b SOFI.Then,the superiority of zero-time-lag auto-cumulant method based on low-pass denoising in high-order SOFI,and increasing spatial sampling frequency by Fourier interpolating are experimentally verified successively.Finally,the experimental results show that the b SOFI can realize a more than 7-fold lateral and axial resolution improvement synchronously,by using zero-time-lag auto-cumulant method based on low-pass denoising after Fourier interpolating,when the order n=10th,numerical aperture NA=0.65,and the fluorescence emission wavelength ?=633nm.Furthermore,a sub-50 nm lateral resolution,amount to a 10.63-fold enhancement,can be obtained only with NA=0.65 by increasing the order to 16 th.The research achievement of this dissertation provides a new method to suppress photonics shot noise and solves the problem of computational difficulty in high-order SOFI,which will promote its application in super-resolution fluorescence microscopy.