Study On Performance Optimization And Application Of Stimulated Emission Depletion Super-resolution Microscopy

Light microscopy is an important tool for human to explore and understand the micro-world.However,the imaging resolution of a light microscope is limited by diffraction,making it impossible to distinguish micro-structures within 200 nm.Super-resolution microscopy technologies were proposed to break the diffraction limit,which improved the resolution of light microscopy by one or two orders of magnitude.As one of a major super-resolution microscopy(SRM)technique,stimulated emission depletion(STED)microscopy was first theoretically proposed and experimentally realized.Under the action of a depletion laser with red-shifted wavelength in STED microscopy,diffraction-unlimited resolution can be obtained by minimizing the point spread function(PSF)via the stimulated emission effect.Compared to other SRM techniques,STED microscopy has the advantages of fast imaging and no post reconstruction needed.However,high depletion laser power and complicated imaging system limit the development and application of STED microscopy.In this thesis,the problems of high depletion laser power and poor imaging quality induced by distorted depletion laser wavefront were addressed.Firstly,for improving the imaging resolution at a lower depletion power,two methods were proposed including increasing fluorescence lifetime to reduce the saturation power and extracting photons with phasor plots analysis.Secondly,for the problem that poor imaging quality induced by distorted laser wavefront,a wavefront compensation approach based on genetic algorithm was proposed.The original depletion laser wavefront was restored to improve the imaging quality and imaging depth of STED microscopy.The main work of this thesis is as follows:1.A home-built pulsed-STED super-resolution imaging system was constructed.Fluorescent beads samples and biological cells samples were prepared to calibrate the optical system,and the lateral resolution reached up to 52 nm and 60 nm,respectively.2.Combined with fluorescence lifetime imaging microscopy,STED imaging system was expanded to a STED-FLIM super-resolution imaging system.The effectof laser power and laser illumination time on the fluorescence lifetime both in confocal and STED imaging mode were investigated.The research results showed that the fluorescence lifetime in confocal imaging mode is independent of the excitation power,while it decreases with the increase of depletion laser power in STED imaging mode.In addition,the fluorescence lifetime increased with the increase of laser illumination time,leading to the reduction of saturated power.Therefore,higher resolution can be obtained at the same depletion laser power.3.The fluorescence signal was collected by this super-resolution fluorescence lifetime imaging system,and the photons were extracted by using phasor plot analysis approach.By using the long-and short-lifetime criterion,the photons with shorter lifetime was removed,and remaining photons carried super-resolution information form an image with improved resolution.4.With the ability of searching local optimal solution by genetic algorithm,an aberration correction program was written for aberration correction of depletion laser beam.In the experiments,the modulation of wavefront phase and the conversion of depletion beam from Gaussian to donut-shaped one were achieved by only one spatial light modulator(SLM),which allowed aberration correction without any additional modification to the original optical system.By using the back-scattering signal of gold nanoparticle as feedback in the process of optimization,the distorted wavefront of depletion beam can be rapidly restored.The research results showed that aberration correction of depletion laser can improve STED imaging quality of thick samples,and super resolution imaging was performed at depths of 24 ?m and 100 ?m in zebrafish retina sample and zebrafish embryo section samples,respectively.The innovative achievements of this thesis primarily reflected in three aspects:1.Based on the STED super-resolution fluorescence lifetime imaging system,it was found that the fluorescence lifetime increased with the laser illumination time.In STED imaging mode,the saturation intensity of the fluorescent probe was reduced by prolonging the laser illumination time,and the resolution was improved at the same depletion laser power.2.Using the ability of phasor plot in analyzing fluorescence lifetime data,acriterion by a line between the central coordinate of STED image and the origin of the coordinate was proposed.The resolution of STED images can be improved at low depletion laser power after extracting the photons with the coordinates closer to the central coordinate of confocal image according to the criterion.3.Genetic algorithm was applied to correct aberration in STED imaging of thick biological samples.After wavefront compensation for depletion laser in the optical system,imaging depths of 24 ?m and 100 ?m were achieved in zebrafish retina sample and zebrafish embryo section samples,respectively.Compared with the images before aberration correction,the signal intensity and resolution of STED images were both improved after aberration correction.