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

加州大学圣克鲁斯分校获得了一项奖励，开发一种用于体内超分辨率成像的自适应光学结构照明显微镜（AO-SIM）。虽然超分辨率成像通过克服远场成像的衍射极限而彻底改变了光学显微镜，但它尚未改变通过厚组织的动态活体成像，这是生物研究界广泛感兴趣的。该项目中AO-SIM的开发是三种不同成像技术的新颖组合，将实现这一目标；结构照明将分辨率从220纳米提高到110纳米，层析成像增加校正体积，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。这种能力将是变革性的，因为实时超分辨率成像只在浅层深度（即1-10微米）得到了证明，而在生物研究界，对更深层的超分辨率成像有明确而广泛的需求。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。将受益于本项目开发的仪器的具体研究实例包括线虫减数分裂期间染色体结构和功能的活体，深层组织体内研究以及果蝇染色质结构和基因表达的调节。这两个研究课题都可以通过仪器的1hz成像速度来解决。仪器开发团队完全有资格开展该项目，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实现了厚（90 ìm）生物组织的实时、动态（0.6秒AO校正时间）衍射极限（220 nm）成像。自适应光学图像校正中用于快速直接波前测量的方法也适用于其他形式的超分辨率和结构照明成像，包括受激发射损耗（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）。仪器开发团队可以访问所需的资源，包括W.M. Keck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），这些资源都位于加州大学圣克鲁斯校区。该项目能够在难以进入的位置提供样品的实时超分辨率图像，为许多生物系统提供新信息。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，这是一所西班牙裔服务机构（HSI）。研究团队成员积极参与帮助维持我们更广泛影响的项目，为有抱负的科学家提供研究经验，重点是为代表性不足的少数民族学生提供服务。这些项目包括美国国家科学基金会资助的加州州立数学与科学暑期学校（COSMOS）项目，科学家与工程师教育研究所（ISEE），以及美国国立卫生研究院资助的少数族裔研究职业和最大化学生发展倡议。COSMOS是一个为期4周的高中学者暑期住宿项目，提供了一个密集的体验，鼓励有前途的年轻学者继续他们对科学和数学的兴趣。COSMOS学生与杰出的研究人员和大学教师并肩工作，涵盖超出典型高中课程的主题。我们的AO显微镜研究成果定期在这些夏季会议上发表，COSMOS的学生在过去的5个夏天都参观了AO显微镜实验室。ISEE还支持新一代科学，技术，工程和数学（STEM）专业人士。该计划围绕三个主要主题：探究，多样性和公平，以及评估。ISEE每年动员当前和未来的STEM专业人员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. Keck自适应光学显微镜中心（CfAOM） （http://cfaom.soe.ucsc.edu/）向用户提供，该中心是一个专注于自适应光学在生物成像中的应用的研究中心。该项目的成果将在生物学家和仪器开发团体参加的会议和讲习班上提出。研究成果将发表在主要期刊、专利申请和CfAOM网站的图片库（iGallery）上。将建立从领先的生物显微镜课程和超分辨率供应商技术网页到iggallery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应用精密公司是通用电气医疗保健公司，该公司表示有兴趣建立商业化合作伙伴关系，一旦AO-SIM成功开发，就可以广泛推广。该奖项由两个项目联合颁发——(1)生物基础设施部（生物科学理事会）的生物研究仪器开发，以及(2)化学部（数学和物理科学理事会）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).