Fast Super-resolution Microscopy Method And Experimental Research Based On Single Molecule Fluorescence Blinking

Recent years,various super-resolution microscopy methodologies have been developed that are capable of imaging features with a resolution well beyond the diffraction limit.These techniques have allowed scientists to study the location and distribution of specific organelles,virus,and protein in fixed cells at a truly molecular-scale resolution level,which has great significance to understand the life process and mechanism of disease.However,when it comes to unveil living cells,due to the intrinsic trade-off between the spatial and temporal resolutions,most existing super resolution microscopy techniques have problems of poor time resolution,high excitation light intensity,and complex system structure,which are not favorable for imaging of live cells,but suitable for fixed cells.Such as the super-resolution localization microscopy(SLM)and super-resolution optical fluctuation imaging(SOFI)techniques,a large number of raw images need to be accumulated to reconstruct one super-resolution image,thus resulting their spatial-temporal resolutions are inadequate to study many dynamic cellular processes at the nanoscale level.This paper aimed at simultaneously improves the spatial-temporal resolutions of SLM and SOFI,thus promoting the development of super-resolution imaging of living cells.Main works in this paper are as follows:1.For superresolution microscopy methods based on single molecule localization,to shorten the exposure time without sacrificing localization precision,it is necessary to maximize the number of photons that can be collected from single molecules per unit time.Here,we describe a novel approach to enhance the signal intensity(collected photons per second)from fluorescence probes by introducing a stimulated emission(SE)optical process to its energy level system.Theoretical results have shown that signal intensity from a single fluorescent molecule can be greatly improved with SE.We therefore showed,using SE in combination with single molecule localization methodology,that fast imaging at a rate of 0.05 s per reconstructed image with lateral resolution of ? 30 nm can be obtained.2.The SOFI technique enhances image spatial resolution by calculating the cumulants of independent stochastic intensity fluctuations of emitters.Its pixel value is not the photons collected from fluorescent molecules,but a statistical analysis of temporal fluctuations.The traditional analysis theory of image signal-to-noise ratio(SNR)does not apply to SOFI technology,which resulting it's unable to provides physical explanation of some experimental phenomenon.Since variance characterizes statistical uncertainty,we determined theoretical expressions for these based on a single dataset.From a simulation of temporal fluctuations of blinking fluorescent emitters,we calculated the quantitative relation between the SNR of cumulants and multiple parameters of the blinking signal,such as the on-time ratio,acquisition frame to average blinking rate ratio,sequence length,and photon amplitude,which not only provides a physical interpretation for SOFI phenomena but also theoretical guidance to achieve optimal practical outcomes.Finally,by simulation using Matlab,we verified that the statistical noise of cumulants dramatically affects SOFI image homogeneities and continuities.3.Based on the statistical analysis theory,the uncertainty of cumulants comes from limited data length will affect the continuity and homogeneity of SOFI image.In traditional SOFI techniques,due to lack of statistical analysis of cumulant,there is no noise constraint condition in the Lucy-Richardson deconvolution to prevent the algorithm from causing noise amplification.In this paper,we calculate the standard deviation in each pixel of SOFI image and introduce the results into the Lucy-Richardson algorithm as a DAMPAR to suppress the noise generation in such pixels.The simulation and experimental results showed that under the same data length,the deconvolution optimization based on standard deviation significantly improves the uniformity and continuity of SOFI image.On the other hand,under the premise of identical image quality,this optimization technique can also greatly shorten the image frames to less than half-tenth of the original,thus improving the SOFI speed 2-10 times.4.Semiconductor photoblinking polymer dots(Pdots)have outstanding photophysical properties,such as high brightness,extraordinary photostability,and favorable biocompatibility,which made it suitable for SOFI imaging with high spatial-temporal resolution.We carried out SOFI imaging of subcellular structures labeled with photoblinking Pdots,which indicates a spatial resolution enhancement of ~2.2-fold with reconstruction from 50 raw images.The main innovation in this thesis is that,a fast super resolution microscopy method has been proposed which combines stimulated emission and single molecule localization technique;Based on the statisticals analysis theory,the quantitative relation between the SNR of cumulants and parameters of blinking signal has been presented;The deconvolution optimization SOFI algorithm has been proposed which greatly shorten the image frames to less than half-tenth of the original,thus improving the imaging speed 2-10 fold.