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

描述（由申请人提供）：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。现有的超分辨成像方法各有优缺点，缺乏一种集单分子灵敏度、大视场、成像速度快和光子毒性小于一体的技术。本研究提出发展一种非线性结构照明显微术（SIM）方法，利用受激发射耗尽（STED）而不是饱和来突破衍射极限。 STED-SIM将实现单分子灵敏度，如单荧光团超分辨率方法，如PALM/STORM。它将在相同的图像速度下将点扫描STED的视场扩大一倍，保持SIM的高动态范围，并达到20~30 nm的分辨率，比饱和结构照明显微镜（SSIM）高2倍。STED-SIM将在全内反射（TIRF）模式下工作，其中倏逝波激发和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、全视场、光毒性小等优点，可为膜驻留或近膜结构的实时超分辨率成像提供更好的解决方案，并可精确跟踪与细胞-基质、细胞-细胞耦合以及近膜细胞相关的问题。 信号在这项研究中，我们将通过两个成像实验证明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.