Ceramic Electron Microscopy Grids for Cell Culturing and Multiscale Imaging

描述（由申请人提供）：需要改进细胞内分子和超分子结构成像的支持技术，以促进细胞生物学研究，并且国立普通医学科学研究所（特别是其细胞生物学和生物物理学部门）正在寻求这种技术。多尺度成像，使用冷冻电子断层扫描（cryo-ET）的超分子结构和单分子，近年来已被证明是一个独特的和宝贵的方法，用于高通量表征的动态三维结构的细胞。电子显微镜（EM）的网格，作为支持生物和生物分子样本被成像的基板，是与这种成像方法相关的关键组成部分。新的EM网格技术降低了样品制备成本和时间，改善了从培养到冷冻的冷冻ET样品的生成，并提高了成像质量，这将使研究人员能够以更高的通量更有效地探索细胞结构。Synkera提出了一种新型陶瓷EM网格，具有集成的薄支撑膜，与细胞培养，光学显微镜和冷冻ET高度兼容。该网格将促进亚细胞架构的高通量、多尺度成像，并提供优于最先进产品的关键优势。该网格也有望成为许多其他EM和培养应用中的竞争性替代品。第一阶段的工作将证明所提出的制造方法的可行性，基于微机械纳米多孔陶瓷。第一阶段还将证明拟议的EM网格与细胞培养，光学显微镜和冷冻EM的兼容性。EM网格将采用两种不同的集成支撑膜选项进行制造。这些网格将与传统的EM网格产品（带有多孔碳薄膜支撑的金网格）进行比较，以证明多尺度细胞和分子成像的更高性能。至少有三个学术合作伙伴将协助展示这些能力。 公共卫生相关性：该项目通过冷冻电子断层扫描（cryo-ET）对分子和细胞进行成像。具体而言，目标应用是通过光学显微镜和细胞，超分子和单分子结构的冷冻ET进行多尺度成像，用于生成亚细胞结构的3D模型。一类新的陶瓷为基础的电子显微镜网格，促进这种多尺度成像的发展提出。所提出的技术将提供比最先进的产品更大的能力，并通过简化标本制备过程和产生上级成像性能来帮助进一步简化多尺度细胞成像。

DESCRIPTION (provided by applicant): Improved supporting technologies for imaging of molecular and supramolecular structures within cells are needed to facilitate cell biology research, and are sought by the National Institute of General Medical Sciences (specifically, its Division of Cell Biology and Biophysics). Multiscale imaging, using cryo-electron tomography (cryo-ET) on supramolecular structures and single molecules, has proven in recent years to be a unique and invaluable method for high-throughput characterization of the dynamic 3D architecture of cells. Electron microscopy (EM) grids, used as substrates for supporting the biological and biomolecular specimens being imaged, are a critical component associated with this imaging method. New EM grid technology that decreases sample preparation cost and time, improves sample generation from culturing to freezing for cryo-ET, and increases imaging quality will allow researchers to more efficiently explore cellular architecture, at higher throughput. Synkera proposes a novel ceramic EM grid that features an integrated thin support film that is highly compatible with cell culturing, light microscopy and cryo-ET. The grids will facilitate high-throughput, multiscale imaging of sub-cellular architecture and offer key advantages over state-of-the art products. The grids are also expected to be a competitive alternative in many other EM and culturing applications. Phase I work will demonstrate feasibility of the proposed fabrication method, based on micromachined nanoporous ceramic. Phase I will also demonstrate compatibility of the proposed EM grids with cell culturing, light microscopy and cryo-EM. EM grids will be fabricated with two different integrated support film options. These grids will be compared to traditional EM grid products (gold grids with holey carbon thin film supports) in order to demonstrate greater performance for multiscale cellular and molecular imaging. At least three academic partners will aide in demonstrating these capabilities. PUBLIC HEALTH RELEVANCE: The project addresses imaging of molecules and cells via cryo-electron tomography (cryo-ET). Specifically, the target application is multiscale imaging via optical microscopy and cryo-ET of cellular, supramolecular and single-molecule structures, for generating 3D models of sub-cellular architecture. The development of a novel class of ceramic-based electron microscopy grids that facilitate this multiscale imaging is proposed. The proposed technology will offer greater capability over state-of-the-art products and help further streamline multiscale cellular imaging by simplifying the specimen preparation process and yielding superior imaging performance.