Super-resolution Full-field Stimulated Emission Depletion Microscope by Structure

描述（申请人提供）：超分辨率荧光显微镜极大地扩展了我们从细胞水平到单分子水平的研究能力。现有的超分辨率方法各有优缺点，缺乏一种能将单分子灵敏度、大视场、快速成像速度和最小光子毒性结合起来的技术。本研究提出了一种非线性结构照明显微镜（SIM）方法，利用受激发射耗尽（STED）代替饱和来突破衍射极限。

DESCRIPTION (provided by applicant): Super resolution fluorescence microscopes greatly expand our ability to study from cellular level down to single molecule level. All existing super-resolution methods have their pros and cons. There lacks a technique that combines single molecule sensitivity, large field of view, fast imaging speed and minimal photon toxicity. This research proposes to develop a nonlinear structured illumination microscopy (SIM) method that utilizes stimulated emission depletion (STED) instead of saturation to break the diffraction limit. STED-SIM will achieve single molecule sensitivity as in single fluorophore super resolution methods, such as PALM/STORM. It will double the field of view of point-scanning STED at the same image speed, retain a high dynamic range as SIM, and reach a resolution of 20~30 nm, a factor of 2 better than saturated structured illumination microscopy (SSIM). STED-SIM will operate at the total internal reflection (TIRF) mode, in which the evanescent excitation and STED field will not cause unnecessary photon toxicity. With its fast speed, high sensitivity, full field of view, and minimal photo toxicity, STED-SIM may provide a better solution for live super-resolution imaging of membrane resident or near membrane structure, and allow precisely following questions related to cell-matrix and cell-to-cell coupling, as well as near membrane cell signaling. In this research we will demonstrated STED- SIM's ability through two imaging experiments: single DNA molecules, and the organization of cell matrix interactions via integrin mediated focal contacts.