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

摘要 这个项目的总体目标是设计一种基因编码的磷光，电子密度高 相关光电子显微镜探测器，或称CLEM，允许单个基因编码 标签将在两种模式下成像，即低温光和电子显微镜。这将大大有助于 在亚纳米、伪原子分辨率的细胞结构建模中，导致了生物医学创新 这取决于在原子水平上对三维细胞结构的理解。在过去的十年里 见证了低温电子显微镜(Cryo-EM)的一场“分辨率革命”，这是由于过去几十年的 显微镜设计、直接电子探测相机、样品制备技术方面的技术进步 和软件开发1。总而言之，这些进展确实给结构生物学领域带来了革命性的变化。 然而，这场革命并不完整。结构研究的最终目的是了解其功能， 大分子在体内的作用机制和动力学。而对分离的络合物的体外研究代表了一种 实现这一目标的关键进展，理想情况下，这些进展应以高分辨率在其 原生蜂窝环境。为此，冷冻电子断层摄影术可以进行三维可视化 细胞结构，尽管分辨率低于单粒子分析2。在该技术中，电池的倾斜系列， 或细胞的切片，是在低剂量条件下获取的。单幅图像的傅里叶变换 然后在倒易空间中给出反投影图像，其中二维序列 然后，变换被组装成细胞结构的单个三维变换。三个人- 然后将细胞的三维倒数空间变换重新投影到实空间，以给出三个- 在低纳米分辨率下的细胞结构的维度视图。使蛋白质在以下位置可视化 在其他细胞蛋白质和细胞器的背景下，我们正在开发一种遗传的 可表达的探头，可同时用于光学显微镜和低温电子断层扫描。这部小说和 创新的探测器将使细胞结构能够被建模为亚纳米分辨率，从而导致生物医学 创新有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.