Establishing a cryogenic correlative light-electron microscopy hub for Oxford

我们要求的设备能让我们以前所未有的细节看到细胞内部。细胞是非凡的、多样的、动态的，它们构成了所有的生物。生物科学研究的一个主要焦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，以及它们在基本过程（如复制）中如何变化。我们还需要了解细胞之间是如何相互作用的，例如，当免疫系统检测到病原体时，或者当神经细胞连接起来产生信号突触时。要做到这一点，我们需要能够观察细胞的结构和内部结构，以及精确定位重要分子的位置，以及它们的位置如何随着细胞的变化和相互作用而改变。电子显微镜可用于提供细胞和生物材料的非常详细的视图，比光学显微镜精确得多。然而，这也带来了挑战。电子显微镜的内部是真空的，生物体无法在真空中生存。因此，在用这种方法研究生物样本之前，有必要仔细制备生物样本。最好的解决方案是冷冻样品，并在整个成像过程中保持在非常寒冷的条件下。这被称为低温电子显微镜，简称cryo-EM。第二个挑战是，由于电子不能穿过细胞，因此细胞太厚，无法在电子显微镜下进行研究。为了解决这个问题，我们制作了薄层，穿过冷冻细胞。这些薄片很薄，可以成像。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。这里，我们要研究的东西用荧光标记，我们可以用荧光显微镜观察细胞，看看标记在哪里。然后我们可以制作细胞的薄层，用荧光信号聚焦在该区域，并在电子显微镜下对其成像。将电子显微镜的高分辨率与荧光标记的靶向检测能力相结合，可以获得更多的信息。在我们的低温电镜设备中，我们已经拥有解决前两个挑战所需的设备，我们正在要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。牛津大学的许多研究人员将使用这个设备来回答各种各样的生物学问题。他们将对神经元相互接触的位置进行成像，并观察当细胞试图找到正确的接触方式时，它们的分子是如何排列的。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中时它们是如何变化的。他们将看到细胞是如何在癌症中出错的过程中移动染色体的。他们将了解细胞内的隔室是如何相互接触和交流的，他们将观察当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以一种新的方式看到细胞内部，发现它们是如何驱动这些以及更多生命所需的过程的。

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.