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。所有现有的超分辨率方法都有其优缺点。目前还缺乏一种结合单分子灵敏度、大视场、快速成像速度和最小光子毒性的技术。本研究建议发展一种利用受激辐射耗竭(STED)而非饱和来突破衍射极限的非线性结构照明显微镜(SIM)方法。 STED-SIM将获得与Palm/Storm等单一荧光团超分辨方法相同的单分子灵敏度。它将在相同的成像速度下将点扫描STED的视场扩大一倍，并保持SIM的高动态范围，分辨率达到20~30 nm，比饱和结构照明显微镜(SSIM)提高2倍。STED-SIM将工作在全内反射(TIRF)模式，在这种模式下，瞬逝的激发和STED场不会引起不必要的光子毒性。STED-SIM具有速度快、灵敏度高、视场大、光毒性小等特点，可为膜残留或膜附近结构的实时超分辨率成像提供更好的解决方案，并允许精确地跟踪与细胞-基质、细胞-细胞耦合以及近膜细胞相关的问题 发信号。在这项研究中，我们将通过两个成像实验来证明STED-SIM的能力：单个DNA分子，以及通过整合素介导的焦点接触来组织细胞基质相互作用。

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.