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

摘要 该项目的总体目标是设计一种基因编码的磷光，电子密度 相关光和电子显微镜（CLEM）的探针，允许单个基因编码 标签在低温光和电子显微镜两种模式下成像。这将大大有助于 在亚纳米、伪原子分辨率下模拟细胞结构， 这取决于在原子水平上理解三维细胞结构。过去十年 在低温电子显微镜（cryo-EM）中见证了“分辨率革命”，这是由于前几十年的 在显微镜设计、直接电子检测照相机、样品制备技术方面的技术进步 软件开发1。结合起来，这些进步确实彻底改变了结构生物学领域。 然而，革命是不完整的。结构研究的最终目标是了解功能， 大分子在体内的作用机制和动力学。虽然分离的复合物的体外研究代表了 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。 原生细胞环境为此，冷冻电子断层扫描允许三维可视化， 细胞结构，尽管分辨率低于单颗粒分析2。在这种技术中，细胞的倾斜系列， 或者说是细胞的切片，是在低剂量条件下拍摄的。单个图像的傅里叶变换 然后，在倒易空间中给出反向投影图像，其中二维序列 然后将这些变换组合成蜂窝结构的单个三维变换。三个- 然后将单元的三维倒易空间变换重新投影回真实的空间，以给出三维倒易空间变换。 在低纳米分辨率下的细胞结构的三维视图。为了使蛋白质能够在 在其他细胞蛋白质和细胞器的背景下，我们正在开发一种遗传学上的超分辨率。 可表达探针，适用于光学显微镜和低温电子断层扫描。这本小说和 创新探针将使细胞结构能够以亚纳米分辨率建模， 创新依赖于在原子水平上理解三维细胞结构。

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.