IDBR: Development and Dissemination of a Flexible Multifunctional Widefield 3D Superresolution Microscopy System for Quantitative Biological Research

摘要此桥接提案解决了生物研究中的一个重要机会，即为美国和全球数千个生物实验室提供多功能3D超分辨率成像能力。该项目的目标是开发一种具有单分子灵敏度的、适用于活细胞研究的突破性多功能显微镜系统，以达到商业化生产的目的。该系统通过提供具有单分子灵敏度的宽视场三维（3D）超分辨率成像，克服了目前的基本限制。这些能力是通过微创荧光技术实现的。智力优势——超分辨率光学显微镜的各种方法现在使得解析小于光学衍射极限的物体成为可能，这在历史上限制了横向尺寸约200nm和轴向尺寸约500nm的空间分辨率。虽然这些技术已经证明了超越衍射极限的光学成像的可行性，但它们还远远不够成熟，需要在典型的生物学实验室中定期达到新的极限。因此，该项目侧重于解决广泛的单分子成像仪器需求的基本发展。该仪器基于照明、荧光分子、3D光学响应、数据收集策略和后处理/重建算法的集成设计。所提出的宽视场显微镜的设计呈现出双螺旋点扩展函数，该函数在像平面上具有两个主导瓣，其角取向随发射器的轴向(z)位置旋转。通过在两个叶的角方向上编码z位置，可以确定每个发射器的三维位置，远远超出光学衍射极限。此外，该技术使3D成像具有比其他成像方法更大的景深。该系统持续实现3D切片能力，各向同性分辨率低于20nm，扩展景深为几微米，比大多数研究机构现有的光学显微镜提高了一个数量级。一个深思熟虑的传播计划提供了从原型开发到大规模生产的路径。该项目的主要开发任务是：(a)将高效双螺旋相位掩模的可扩展制造工艺推向成熟。(b)推进重建算法的实施，以方便生物学家实时操作和使用。(c)采用可与新的或现有显微镜结合使用的灵活模块化结构。(d)在重大生物问题和独立生物实验室中测试仪器。更广泛的影响——3D超分辨率成像的广泛应用将影响科学和工程的多个领域，包括细胞内外标记生物分子的3D生物物理和生物医学成像。该技术将用于跟踪细胞结构内分子的取向变化，以及监测分子之间的相互作用。该项目将教育和推广与研究和开发相结合，为开发未来生物仪器技术创造所需的人类基础设施。它将为各种各样的年轻科学家和工程师提供培训。通过与当地计算光学传感和成像的IGERT项目的互动，该项目将为跨学科的学生轮岗提供机会。该项目还将与工业界建立协同关系，将新发现转化为工业应用。调查结果将广泛传播，包括一个外联巡回展览显微镜与互动示范。

AbstractThis bridging proposal addresses a major opportunity in biological research to provide multifunctional 3D superresolution imaging capability to thousands of biology laboratories in the US and around the globe. The objective of this project is to develop, to the point of commercial production, a ground-breaking multifunctional microscopy system with single-molecule sensitivity suitable for live cell studies. The system overcomes current fundamental limitations by providing wide-field three-dimensional (3D) super-resolution imaging with single molecule sensitivity. These capabilities are achieved using minimally invasive fluorescence techniques.Intellectual merit--A variety of methods for super-resolution optical microscopy are now making it possible to resolve objects that are smaller than the optical diffraction limit, which has historically restricted spatial resolution to about 200nm in the transverse dimension and about 500 nm in the axial dimension. Although these techniques have demonstrated the feasibility of optical imaging beyond the diffraction limit, they are far from mature and new developments are required to reach the new limits on a regular basis at the typical biology lab.Accordingly, this project focuses on fundamental developments to address the need for widespread single-molecule imaging instrumentation. The instrument is based on an integrated design of the illumination, the fluorescent molecules, the 3D optical response, the data collection strategy, and the postprocessing / reconstruction algorithms.The proposed design of a wide-field microscope presents a double-helix point spread function that features two dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. By encoding the z-position in the angular orientation of the two lobes, the 3D position of each emitter can be determined well beyond the optical diffraction limit. Moreover, the technique enables 3D imaging with greater depth of field than is available from other imaging methods. The system consistently attains 3D sectioning capability with isotropic resolutions below 20nm over an extended depth of field of several microns, representing one order of magnitude improvement over available optical microscopes at most research institutions.A well thought-out dissemination plan provides a path from prototype development through large scale production. The main development tasks in this project are: (a) Bringing to maturity a scalable fabrication process for efficient double-helix phase masks. (b) Advancing the implementation of reconstruction algorithms for real-time operation and ease of use by biologists. (c) Implementing a flexible modular structure that can be integrated with new or existing microscopes. (d) Testing the instrument in significant biological problems and independent biology labs.Broader Impact - The widespread availability of 3D superresolution imaging will impact multiple fields of science and engineering including 3D biophysical and biomedical imaging of labeled biomolecules inside and outside of cells. The techniques will find use in tracking orientation changes of molecules within a cellular structure, as well as in monitoring interactions between molecules.The project integrates education and outreach with research and development to create the human infrastructure required for developing future biological instrumentation technologies. It will provide training to a diverse group of young scientists and engineers. Through interaction with a local IGERT program in computational optical sensing and imaging, this project will provide opportunities for interdisciplinary student rotations. The project will also generate synergistic relationships with industry to transfer new discoveries into industrial applications. The results of the investigation will be broadly disseminated including an outreach traveling exhibit on microscopy with interactive demonstrations.