Establishing a cryogenic correlative light-electron microscopy hub for Oxford

为牛津建立低温关联光电子显微镜中心

• 批准号: BB/X019276/1
• 负责人: Matthew Higgins
• 金额: $ 75.97万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX019276%2F1
• 关键词: Establishing; cryogenic; correlative; light; electron

We are asking for equipment which will allow us to see inside cells in unprecedented detail.Cells are extraordinary, varied and dynamic and they make up all living things. A major focus of biosciences research is to understand the varying structures of cells, allowing us to understand how they are constructed and how they change during fundamental processes, such as replication. We also need to understand how cells interact with each other, for example as the immune system detects a pathogen or as nerve cells connect to create a signalling synapse. To do this, we need to be able to observe the architecture and internal structures of cells, as well as to pin-point the locations of important molecules, and how their positions alter as cells change and interact.Electron microscopes can be used to provide highly detailed views of cells and biological material and are much more precise than light microscopes. However, this brings challenges. The inside of an electron microscope is in a vacuum, in which living organisms cannot survive. It is therefore necessary to prepare a biological sample carefully before it can be studied in this way. The best solution is to freeze the sample and to maintain it in very cold conditions throughout imaging. This is called cryogenic electron microscopy, or cryo-EM. A second challenge is that cells are too thick to study in an electron microscope as the electrons cannot pass through a cell. To solve this, we make thin layers, which cut through frozen cells. These lamella are thin enough to image. Finally, cells are large and complex and the images taken on electron microscopes are therefore crowded. It can be hard to find what we want to look at. To solve this, we can use a technique called correlative light-electron microscopy (cryo-CLEM). Here, the things which we want to study are labelled using a fluorescent marker and we can observe the cells using a fluorescence microscope to see where the marker is. We can then make thin layers of the cell, focusing in on the region with the fluorescence signal and can image them in an electron microscope. By correlating the images from the fluorescence and electron microscope we can get much more information, combining the higher resolution of the electron microscopy with the targeted detected capability which comes from fluorescence labelling. Within our cryo-EM facility, we already have the equipment required to solve the first two of these challenges and we are asking for the microscope and associated equipment to allow us to conduct fluorescence microscopy under cryogenic conditions. This equipment will be used by many researchers from across Oxford, to answer all kinds of questions about biology. They will image the sites where neurons contact each other and see how their molecules arrange as the cells are trying to find their way to make the right contacts. They will image the genomes of bacteria and see how they change when the bacteria are exposed to antibiotics. They will see how cells move their chromosomes around in processes which go wrong in cancer. They will understand how the compartments within cells contact and communicate with one another and they will observe what happens when parasites contact human cells and when immune cells contact pathogens. The capability provided by cryo-CLEM will allow us to see inside cells in a new way, to discover how they drive these, and many more, processes needed for life.

我们要求的设备将使我们能够看到细胞内部前所未有的细节。细胞是非凡的，多样的和动态的，它们构成了所有的生物。生物科学研究的一个主要重点是了解细胞的不同结构，使我们能够了解它们是如何构建的，以及它们在复制等基本过程中是如何变化的。我们还需要了解细胞如何相互作用，例如，当免疫系统检测到病原体或神经细胞连接以创建信号突触时。为了做到这一点，我们需要能够观察细胞的结构和内部结构，以及确定重要分子的位置，以及它们的位置如何随着细胞的变化和相互作用而变化。电子显微镜可以用来提供细胞和生物材料的高度详细的视图，并且比光学显微镜精确得多。然而，这带来了挑战。电子显微镜的内部处于真空状态，生物体无法在其中生存。因此，在以这种方式进行研究之前，有必要仔细制备生物样品。最好的解决方案是冷冻样品，并在整个成像过程中将其保持在非常冷的条件下。这就是所谓的低温电子显微镜，或cryo-EM。第二个挑战是细胞太厚，无法在电子显微镜下进行研究，因为电子无法通过细胞。为了解决这个问题，我们制作了薄层，可以切开冷冻的细胞。这些薄片薄到足以成像。最后，细胞又大又复杂，因此在电子显微镜上拍摄的图像很拥挤。很难找到我们想看的东西。为了解决这个问题，我们可以使用一种称为相关光电子显微镜（cryo-CLEM）的技术。在这里，我们要研究的东西是用荧光标记物标记的，我们可以用荧光显微镜观察细胞，看看标记物在哪里。然后，我们可以制作细胞的薄层，聚焦在具有荧光信号的区域，并可以在电子显微镜下对其成像。通过将来自荧光和电子显微镜的图像相关联，我们可以获得更多的信息，将电子显微镜的更高分辨率与来自荧光标记的靶向检测能力相结合。在我们的cryo-EM设施中，我们已经拥有解决前两个挑战所需的设备，我们正在寻求显微镜和相关设备，使我们能够在低温条件下进行荧光显微镜检查。牛津大学的许多研究人员将使用这种设备来回答各种生物学问题。他们将对神经元相互接触的部位进行成像，并观察它们的分子如何排列，因为细胞试图找到正确的接触方式。他们将对细菌的基因组进行成像，并观察当细菌暴露于抗生素时它们如何变化。他们将看到细胞如何在癌症中出错的过程中移动染色体。他们将了解细胞内的隔室如何相互接触和交流，他们将观察寄生虫接触人体细胞和免疫细胞接触病原体时会发生什么。cryo-CLEM提供的能力将使我们能够以一种新的方式看到细胞内部，发现它们如何驱动这些以及更多生命所需的过程。