A Genetically Encoded Phosphorescent, Electron Dense Probe for Correlative Light and Electron Microscopy

用于相关光和电子显微镜的基因编码磷光电子致密探针

• 批准号: 10547694
• 负责人: CHARLES MARTIN LAWRENCE
• 金额: $ 29.92万
• 依托单位: PHOTON BIOSCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10547694
• 关键词: 3-Dimensional; Algorithms; Back; Beds; Binding; Biological Sciences; Caliber; Cell model; Cells; Cellular Structures; Chimeric Proteins; Complex; Computer software; Cryo-electron tomography; Cryoelectron Microscopy; Data; Dose; Electron Microscopy; Electrons; Engineering; Environment; Escherichia coli; Fluorescence; Fourier Transform; Freezing; Fusion Protein Expression; Goals; Image; In Vitro; Individual; Ions; Lanthanoid Series Elements; Location; Medical; Microscope; Modality; Modeling; Modernization; Molecular; Montana; Organelles; Photons; Positioning Attribute; Preparation; Proteins; Protocols documentation; Pseudomonas aeruginosa; Research Personnel; Resolution; Sampling; Series; Slice; Structure; Techniques; Technology; Testing; Thick; Time; Tomogram; Universities; Visualization; Work; cell transformation; cryogenics; density; design; electron tomography; in vivo; innovation; interest; light microscopy; macromolecule; nanometer; nanometer resolution; novel; particle; phosphorescence; software development; structural biology; three-dimensional visualization; tomography; two-dimensional

Abstract The overall goal of this project is to engineer a genetically encoded phosphorescent, electron dense probe for Correlative Light and Electron Microscopy, or CLEM, that allows a single genetically encoded tag to be imaged under both modalities, cryogenic light and electron microscopy. This would greatly aid in modeling cellular structures at sub-nanometer, pseudo-atomic resolution, leading to biomedical innovations dependent upon understanding 3-dimensional cellular structures at the atomic level. The last decade has witnessed a “Resolution Revolution” in cryo-electron microscopy (cryo-EM) due to preceding decades of technical advancement in microscope design, direct electron detecting cameras, sample preparation techniques and software development1. Combined, these advances have indeed revolutionized the field of structural biology. However, the revolution is incomplete. The ultimate goal of structural studies is to understand the function, mechanism and dynamics of macromolecules in vivo. While in vitro studies of isolated complexes represents a critical progress towards this goal, ideally they should be visualized at high resolution within the context of their native cellular environments. To this end, cryo-electron tomography allows three-dimensional visualization of cellular structures, albeit at lower resolution than single particle analysis2. In this technique a tilt series of the cell, or a slice through the cell, is taken under low dose conditions. The Fourier transforms of the individual images are then taken to give the back projected image in reciprocal space, where the series of two-dimensional transforms are then assembled into a single three-dimensional transform of the cellular structure. The three- dimensional reciprocal space transform of the cell is then re-projected back into real space to give a three- dimensional view of the cellular structure at low nanometer resolution. To enable proteins to be visualized at super-resolution within the context of other cellular proteins and organelles, we are developing a genetically expressible probe that works for both light microscopy and cryogenic electron tomography. This novel and innovative probe will enable cellular structures to be modeled sub-nanometer resolution, leading to biomedical innovations dependent upon understanding 3-dimensional cellular structures at the atomic level.

抽象的 该项目的总体目标是设计一种基因编码的磷光、电子密集型 用于相关光电子显微镜（CLEM）的探针，允许单个基因编码 标签在低温光学和电子显微镜下成像，这将有很大帮助。 以亚纳米、伪原子分辨率模拟细胞结构，从而带来生物医学创新 依赖于在原子水平上理解 3 维细胞结构。 由于前几十年的研究，冷冻电子显微镜 (cryo-EM) 经历了一场“分辨率革命” 显微镜设计、直接电子检测相机、样品制备技术方面的技术进步 结合起来，这些进步确实彻底改变了结构生物学领域。 然而，结构研究的最终目标是理解功能， 体内大分子的机制和动力学，而分离复合物的体外研究代表了一个。 实现这一目标的关键进展，理想情况下，它们应该在其背景下以高分辨率可视化 为此，冷冻电子断层扫描可以实现细胞的三维可视化。 细胞结构，尽管分辨率低于单粒子分析2。在该技术中，细胞的倾斜系列， 或细胞切片，是在低剂量条件下拍摄的单个图像的傅里叶变换。 然后给出倒易空间中的反投影图像，其中一系列二维 然后将变换组合成细胞结构的单个三维变换。 然后将细胞的维度倒易空间变换重新投影回真实空间以给出三- 以低纳米分辨率观察细胞结构的三维视图。 在其他细胞蛋白质和细胞器的背景下进行超分辨率，我们正在开发一种遗传方法 这种新颖且适用于光学显微镜和低温电子断层扫描的可表达探针。 创新探针将使细胞结构能够以亚纳米分辨率建模，从而实现生物医学 创新依赖于在原子水平上理解 3 维细胞结构。