Research On Wide-field Fluorescence Super-resolution Microscopy

In recent years, fluorescence super-resolution imaging system has been greatly developed. The availability of breaking the diffraction limited and noninvasive has made it one of the most successful techniques in the biological and medical imaging. In this paper we have introduced a new super-resolution imaging technique based on the stochastic photobleaching. This article introduces the basic principle of this technique by combined the following aspects:the production mechanism of the fluorescence, fluorescent molecules, the basic principle and algorithm of the single molecule localization, photobleaching phenomenon. This technique relies on intrinsic and stochastic bleaching of fluorescent probes. We simply acquire a stream of fluorescence images, and single fluorophores are detected by subtracting each frame from the previous one. After image subtractions, we can get a super-resolution image by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them.In generally we used wide field illumination in the fluorescence microscopy, for it has a wide field imaging field, and needs less time. But due to the presence of high background noise it will affect the final image. So in this paper we present an improved method, evanescent wave fields generated by the internal reflection are used to illuminate the sample, and only fluorophores within a thickness of 100 nm thick from the surface are activated, improving the signal-to-noise ratio (SNR) of the image. This paper simulates the method and verifies feasibility of the method. Discuss the influence of signal-to-noise ratio, picture number, the average fluorescence to the final image quality. Finally, we design and set up the experimental system, sub-diffraction imaging with wide-filed illumination and TIRF microscopy by stochastic photobleaching is studied. This method can yield contrast-enhanced images with a higher SNR and improve the lateral resolution.