Energy filter with direct electron detector for electron cryo tomography

生命机器在分子和原子水平上在生物细胞和组织中运行。这些分子机器的详细描述，它们的内部组织，以及它们在细胞内的相互作用是了解健康和疾病的生物体的基础。 在伯克贝克，我们研究这些机器的三维结构，目的是了解它们是如何工作的。电子显微镜（EM）已成为观察生物机器的主要技术，无论是在隔离和在他们的细胞环境。电子显微镜可以提供关于生物分子结构的非常详细的信息，以及关于细胞和组织组织的整体观点。使我们能够确定细胞机器的三维结构的EM方法是电子断层扫描。从原理上讲，这与医学上的计算机断层扫描非常相似，只是它的放大倍数非常高，给我们显示的是细胞和分子，而不是骨骼、血管和器官。样本使用低温液体快速冷冻，在非常低的温度下进行检查。使用这种类型的制剂，它们可以在其自然的水合状态下成像。在过去的十年中，电子显微镜方法的进步使我们能够获得生物分子在其正常运作的不同阶段的快照，揭示其作用机制的细节。然而，在其自然环境中快速冷冻而捕获的未染色生物结构对电子的散射非常弱，并且对电子束高度敏感。所得到的图像具有差的对比度和高背景，限制了图像中可检测的结构特征。在这项设备补助金中，我们正在申请高分辨率电子能量过滤器和电子探测器的资金，以帮助我们记录细胞断层扫描图像中的精细结构细节。该过滤器消除了电子通过细胞厚的样品时产生的不必要的非弹性散射。过滤掉这种类型的散射使图像更清晰，以便可以识别和解释更多的结构细节。这种能量过滤器将使我们能够提取更多关于细胞机器如何运作的信息。将受益于这种新设备的具体项目包括：1）研究病原体感染的细胞（包括疟疾、衣原体和弓形虫），将揭示这些细胞内入侵者如何破坏宿主细胞系统; 2）研究细菌分泌系统，揭示细菌如何与环境相互作用，以促进自身的生存和复制; 3）研究病毒感染细菌的过程; 4）研究我们的免疫细胞如何保护我们的身体免受感染或癌症; 5）研究神经元细胞的结构和微管细胞骨架，这将揭示它们如何在大脑发育过程中适应它们的形状，以及如何在人类疾病中被破坏; 6）研究淀粉样蛋白在活细胞中的沉积，这将揭示细胞的质量控制机制如何处理导致神经退行性疾病的蛋白质错误折叠。

The machinery of life operates in biological cells and tissues at the molecular and atomic level. A detailed description of these molecular machines, their internal organization, and their interactions within cells is fundamental to understanding living organisms in health and disease. At Birkbeck we study the three-dimensional structure of these machines with the aim of understanding how they work. Electron microscopy (EM) has become a major technique for observing biological machines both in isolation and in their cellular context. Electron microscopy can provide highly detailed information on the structure of biological molecules as well as overall perspectives on the organization of cells and tissues. The EM approach that allows us to determine the three-dimensional structures of cellular machinery is electron tomography. In principle, this is very similar to computed tomography in medicine, except that it works at very high magnification and shows us cells and molecules rather than bones, blood vessels and organs.The samples are rapidly frozen using cryogenic liquids and examined at very low temperature. With this type of preparation they can be imaged in their natural, hydrated state. Advances in electron microscopy methods in the last decade have enabled us to obtain snapshots of biological molecules at different stages of their normal operation, revealing details of their mechanisms of action. However, the unstained biological structures trapped by rapid freezing in their native environment scatter electrons very weakly and are highly sensitive to the electron beam. The resulting images have poor contrast and a high background, limiting the detectable structural features in the images. In this equipment grant, we are requesting funding for a high-resolution electron energy filter and electron detector to help us record fine structural details in tomography images of cells. The filter removes unwanted, inelastic scattering that arises when electrons pass through samples as thick as cells. Filtering out this type of scattering makes the images clearer, so that more structural details can be discerned and interpreted. This energy filter will enable us to extract more information about how the cellular machinery functions.Specific projects that will benefit from this new equipment include: 1) studies of pathogen-infected cells (including Malaria, Chlamydia and Toxoplasma) that will reveal how these intracellular invaders sabotage host cell systems; 2) studies of bacterial secretion systems that will reveal how bacteria interact with their environment to promote their own survival and replication; 3) studies of the process by which viruses infect bacteria; 4) studies to reveal how our immune cells protect our bodies from infection or cancer; 5) studies of the architecture and microtubule cytoskeleton of neuronal cells that will reveal how they adapt their shape during brain development and how this is disrupted in human diseases; 6) studies of amyloid protein deposits in living cells that will reveal how the cell's quality control machinery handles protein misfolding that causes neurodegenerative disease.