IDBR: Type A Adaptive Optical Structured Illumination Microscope (AOSIM)

加州大学圣克鲁斯分校被授予开发一种用于活体超分辨率成像的自适应光学结构照明显微镜(AO-SIM)。虽然超分辨率成像克服了远场成像中的衍射限制，使光学显微镜发生了革命性的变化，但它还没有通过厚组织来改变动态活体成像，这引起了生物研究界的广泛兴趣。本项目中开发的AO-SIM是三种不同成像技术的新颖组合，将使这一点成为可能：结构照明将分辨率从220 nm提高到110 nm，断层摄影术可以增加校正体积，以及低音高音自适应光学系统足以使校正深度增加一个数量级。这一能力将是变革性的，因为现场超分辨率成像仅在浅层(即1-10微米)进行了演示，在生物研究界的更深层对其有明显和广泛的需求。使用超分辨率显微镜的活跃研究领域包括细胞结构、膜异质性和动态蛋白质组装。将从该项目开发的仪器中受益的研究的具体例子包括对线虫减数分裂过程中染色体结构和功能的活体深层组织研究，以及对果蝇染色质结构和基因表达的调节。这两个研究主题都可以用仪器的1赫兹成像速度来解决。仪器开发团队完全有资格实施该项目，因为他们之前开发了第一台自适应光学显微镜，该显微镜使用直接波前传感在厚(90？m)生物组织中实现实时、动态(0.6秒AO校正时间)衍射限制(220 Nm)成像。用于自适应光学图像校正的快速直接波前测量方法也适用于其他形式的超分辨率和结构照明成像，包括受激发射耗尽(STED)显微镜、光激活定位显微镜(Palm)、随机光学重建显微镜(STORM)和选择性平面成像显微镜(SPIM)。仪器开发团队可以使用所需的资源，包括W.M.凯克自适应光学显微镜中心(CfAOM)、加州大学自适应光学中心(CFAO)和UCO/Lick自适应光学实验室(LAO)，所有这些都位于加州大学圣克鲁斯分校。该项目能够在难以接近的地点现场拍摄样本的超分辨率图像，为许多生物系统提供新的信息。它还为加州大学圣克鲁斯分校的学生提供尖端光学科学培训，这是一家为拉美裔服务的机构(HSI)。研究团队成员是项目的积极参与者，这些项目有助于维持我们更广泛的影响，为有抱负的科学家提供研究经验，重点是服务于代表不足的少数族裔学生。其中包括NSF资助的加州州立数学与科学暑期学校(COSMOS)计划、科学家与工程教育工作者研究所(ISEE)，以及NIH资助的少数族裔获得研究职业机会和最大限度促进学生发展的倡议。COSMOS是一个为高中学者提供的为期4周的暑期住宿项目，它提供了密集的体验，鼓励有前途的年轻学者继续对科学和数学感兴趣。COSMOS的学生与杰出的研究人员和大学教职员工并肩工作，涵盖了超出典型高中课程的主题。我们的声光显微镜研究成果定期在这些暑期会议上公布，在过去的5个夏天里，COSMOS的学生一直在参观声光显微镜实验室。ISEE还支持新一代科学、技术、工程和数学(STEM)专业人员。该计划围绕三个主要主题构建：调查、多样性和公平以及评估。ISEE动员现在和未来的STEM专业人员每年教授和指导数百名本科生和研究生。由此产生的下一代仪器将在W.M.凯克自适应光学显微镜中心(http://cfaom.soe.ucsc.edu/)，)向用户提供，该中心专注于自适应光学在生物成像中的应用。该项目的成果将在生物学家和仪器开发界参加的会议和讲习班上介绍。研究结果将发表在领先的期刊、专利申请和CfAOM网站的图片库(IGallery)上。将从领先的生物显微镜课程和超分辨率供应商技术网页建立到iGallery的网络链接，以便向生物研究界宣传新的成像能力。结构照明显微镜的原始供应商，通用电气医疗保健公司旗下的应用精密公司，已经表示有兴趣建立商业化合作伙伴关系，在成功开发后广泛传播AO-SIM。该奖项由两个项目联合颁发-(1)生物基础设施司(生物科学局)的生物研究仪器开发，以及(2)化学部(数学和物理科学局)的化学测量和成像。

An award is made to the University of California Santa Cruz to develop an Adaptive Optical Structured Illumination Microscope (AO-SIM) for in-vivo super-resolution imaging. While super-resolution imaging has revolutionized optical microscopy by overcoming the diffraction limit in far-field imaging, it has yet to transform dynamic live in vivo imaging through thick tissue, which is of broad interest to the biological research community. The development of an AO-SIM in this project is a novel combination of three different imaging techniques that will enable this; structured-illumination to double the resolution from 220 nm to 110 nm, tomography to increase the corrected volume, and a woofer-tweeter adaptive optical system sufficient for an order of magnitude increase in the depth of correction. This capability will be transformative since live super-resolution imaging has only been demonstrated at superficial depths (i.e. 1-10 microns), and there is a clear and broad need for it at deeper depths within the biological research community. Active areas of research using super-resolution microscopy include cellular architecture, membrane heterogeneity and dynamic protein assembly. Specific examples of research that will benefit from the instrument developed in this project include live, deep-tissue in vivo studies of chromosome structure and function during meiosis in C. elegans and the regulation of chromatin structure and gene expression in Drosophila. Both of these research topics can be addressed with the 1 Hz imaging speed targeted for the instrument. The instrument development team is well qualified to conduct the project as they have previously developed the first adaptive optical microscope that used direct wavefront sensing to achieve live, dynamic (0.6 sec AO correction time) diffraction limited (220 nm) imaging in thick (90 ìm) biological tissues. The approach used for fast direct wavefront measurement in adaptive optical image correction is also applicable to other forms of super-resolution and structured illumination imaging including Stimulated Emission Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM), and Selective Plane Imaging Microscopy (SPIM). The instrument development team has access to the required resources including the W.M. Keck Center for Adaptive Optical Microscopy (CfAOM), the UC Center for Adaptive Optics (CfAO), and the UCO/Lick Laboratory for Adaptive Optics (LAO), all located on the UC Santa Cruz campus. This project enables live super-resolution images of samples in difficult to access locations providing new information for a host of biosystems. It also provides training in cutting edge optical science for students at UC Santa Cruz, a Hispanic-Serving Institution (HSI). The research team members are active participants in programs that help sustain our broader impact, providing research experiences to aspiring scientists, with emphasis on serving underrepresented minority students. These include the NSF-funded California State Summer School for Mathematics and Science (COSMOS) program, the Institute for Scientist & Engineer Educators (ISEE), and the NIH-funded Minority Access to Research Careers and Initiative for Maximizing Student Development. COSMOS, a 4-week summer residential program for high school scholars, provides an intensive experience that encourages promising young scholars to continue their interest in science and mathematics. COSMOS students work side-by- side with outstanding researchers and university faculty, covering topics that extend beyond the typical high school curriculum. Our AO microscopy research results are presented regularly at these summer sessions and COSMOS students have been visiting the AO microscopy lab for the last 5 summers. ISEE also supports a new generation of science, technology, engineering and mathematics (STEM) professionals. The program is structured around three major themes: inquiry, diversity and equity, and assessment. ISEE mobilizes current and future STEM professionals to teach and mentor hundreds undergraduates and postgraduates each year. The resulting next-generation instrument will be made available for users at the W.M. Keck Center for Adaptive Optical Microscopy (CfAOM) (http://cfaom.soe.ucsc.edu/), a research center focused on the application of adaptive optics in biological imaging. The results of the project will be presented at conferences and workshops attended by both biologists and instrument development communities. The research results will be published in leading journals, patent applications and in an image gallery (iGallery) on the CfAOM website. Web links will be established to the iGallery from the leading biological microscopy courses and super-resolution vendor technology web pages in order to advertise the new imaging capability to the biological research community. The original vendor of structured illumination microscopes, Applied Precision, a GE Healthcare Company, has expressed an interest in a commercialization partnership to broadly disseminate the AO-SIM once it is successfully developed. This award is being made jointly by two Programs- (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Chemical Measurements and Imaging, in the Division of Chemistry (Math and Physical Sciences Directorate).