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遗传编码的磷光，电子密集 相关光和电子显微镜或CLEM的探针，该探针允许单个一般编码 标签将在两种方式，低温光和电子显微镜下成像。这将有很大的帮助 在对伪原子分辨率下的细胞结构建模时，导致生物医​​学创新 取决于理解原子水平的3维细胞结构。过去十年有 由于前几十年 显微镜设计的技术进步，直接电子检测相机，样品制备技术 和软件开发1。这些进步的结合确实彻底改变了结构生物学领域。 但是，革命是不完整的。结构研究的最终目标是了解该功能， 体内大分子的机理和动力学。而在体外研究分离的复合物代表 朝着这一目标的关键进展，理想情况下，应在他们的背景下以高分辨率可视化它们 天然细胞环境。为此，冷冻电子层析成像允许三维可视化 细胞结构，尽管分辨率低于单个粒子分析2。在此技术中，一个倾斜的单元系列 或通过细胞切片在低剂量条件下采取。单个图像的傅立叶变换 然后将其用于在互惠空间中给出背面的图像，其中一系列二维 然后将变换组装成细胞结构的单个三维变换。三 然后将细胞的尺寸相互空间变换重新投影回真实空间，以提供三 低纳米分辨率下细胞结构的尺寸视图。使蛋白质能够可视化 在其他细胞蛋白质和细胞器的背景下，我们正在开发一个普遍的 表达探针，可用于光学显微镜和低温电子断层扫描。这本小说和 创新的探针将使细胞结构能够建模为子纳米分辨率，从而导致生物医​​学 创新取决于理解原子水平的3维细胞结构。