Implementation And Application Of DsTORM Super-Resolution Imaging

Fluorescence microscopy has become one of the most powerful and essential tools for biological studies because of its relatively noninvasive and molecularly specific. However, the spatial resolution of optical microscopies has an inherent limitation due to the light diffraction. According to Rayleigh criterion, it is limited to 200nm in lateral dimension and 500nm in axial dimension, which can’t discern the details of biological structures. Over the past years, several methodologies have been developed for super-resolution fluorescence microscopy to break the diffraction limit, such as Stimulated Emission Depletion Microscopy (STED), Photoactivated Localization Microscopy(PALM), (direct)Stochastic Optical Reconstruction Microscopy (dSTORM/STORM).In this study, a super-resolution imaging platform based on single molecular imaging was built with our available experimental equipment. With this system, we made PALM and (d)STORM come true and achieved the super-resolution images of actin filaments and microtubules. Meanwhile, we also observed a high resolution of p-body. Results were as follows:(1)The system of the super-resolution imaging based on single-molecule localization was established. It consists of a Nikon inverted microscope with an oil immersion TIRF objective, an AOTF system to control the lasers, an electron multiplying charged-coupled device camera to capture images and so on. Besides of this, we completed the program of single molecular localization algorithms with Matlab software and verified its feasibility.(2)Then we performed on the imaging system to acquire super-resolution imaging of microtubules and actin filaments using PALM or dSTORM with suitable devices. We performed dSTORM to imaging actin filaments and microtubules labeled with Photoswitchable dye Alexa Fluor 647 and performed PALM to imaging actin filaments labeled with Photoactivated protein Dendra2. Dendra2-actin was transfected into Hela cells for imaging. As a result, we acquire more details of subcellular structures than conventional fluorescence microscopy.(3)After that, we had focused on the optimization of experimental conditions and imaging parameters for dSTORM. We varied the concentration of switching buffer to promote the florescent blinking and optimized the protocols to improve the imaging resolution. Finally, we had got the optimum conditions for dSTORM imaging and achieved a high resolution of 24 nm in in lateral dimension.(4)Finally, a super-resolution image of P-bodies, labeled with Alexa Fluor 647, was constructed for single molecular imaging based on the principle of dSTORM. Compared to conventional fluorescence images, it showed the advantage to resolve the subcellular events and could be used to explore the relationship between p-bodies and miRNAs.