Research On Methods And Techniques Of Imaging Signal-to-noise Ratio And Contrast Enhancement In STED Super-resolution Microscopy

Far-field fluorescence optical microscopy plays an important role in biomedical researches due to its low damage to biological tissues,specific labeling and in vivo imaging.It is an important tool for people to understand the microscopic world and conduct scientific research.However,due to the limitation of diffraction limit,the resolution of traditional far-field optical microscopy is restricted to about half the wavelength,which cannot achieve effective observation of the internal structure of cells and other samples.In order to break through this bottleneck,super-resolution microscopy(or called nanoscopy)came into being and has made great progress in the past two decades.As one of the mainstream methods in super-resolution microscopy,stimulated emission depletion(STED)microscopy occupies an important position in this field.It can achieve imaging resolution much higher than the diffraction limit and has been applied to practical researches in some field like biomedicine.However,current STED technique still has the problem of high system complexity,high depletion intensity and subsequent photobleaching,signal-to-noise ratio degradation and etc.Also,background signals generated by excitation light and depletion light will also affect the actual imaging contrast and imaging quality.This thesis focuses on STED microscopy,studying its system design and system implementation.By means of new detection methods and time-domain and frequency-domain processing to suppress the influence of background signals,exploiting the enhancement methods towards imaging SNR and contrast,thus improving the resolving ability and imaging quality.Specifically,this thesis designs and implements an integrated dual-color STED system.Next,through array detection and photon reassignment methods,we propose and implement parallel detection STED microscopy,which can improve the SNR and resolution of traditional STED imaging.In addition,we proposed and implemented the time-domain double modulation STED method and system,which can effectively decrease the influence of the background signal in STED and improve the imaging contrast and resolution.Also,the current main STED contrast enhancement methods are analyzed,and the advantages and disadvantages of each technique are also compared and summarized.The main innovations of this thesis are as follows1.STED based on parallel detection is proposed for the first time.Aiming at the detection efficiency bottleneck caused by the saturation effect in traditional single-point detectors,the array detector consists of specific fiber bundles and avalanche photodiode(APD)arrays effectively improves the detection efficiency of the STED system.Subsequently,combined with photon reassignment algorithm to process the array detection images,2-5 times SNR enhancement can be achieved and the imaging resolution are also further improved.2.STED combined time-domain double modulation methods is proposed,which can suppress the influence of the background signal in traditional STED and improve the imaging resolution and contrast.Using EOM,AOTF modulation and lock-in amplifier demodulation,a time-domain double modulation STED system is realized,and the effect of improving imaging contrast and resolution is confirmed through experiments.