Equipment for Correlative LM/EM by High Pressure Freezing and Tomography

描述(申请人提供)：近些年来透射式电子显微镜最重要的发展之一是用于自动电子断层扫描的仪器。电子层析成像(ET)产生高分辨率的细胞成分的3D图像，有助于弥合分子结构生物学和活体细胞生物学结构研究之间的差距。在一些断层摄影术研究中，实际上有可能在细胞的三维模型中识别分子的特征结构。需要资金用于为Tecnai 12升级现有的Tecnai 12生物双胞胎电子显微镜的Xplore 3D改装套装，用于自动断层扫描数据收集，以及Leica EMPACT2高压冷冻机和Live Cell成像系统，以允许在光学显微镜中查看活细胞，然后在大约5秒内将其冷冻。这套仪器系统将大大提高可获得的电子断层扫描数据的质量和数量。细胞将在光学显微镜中被活着观察，当它们达到细胞周期或发育的正确阶段时，它们将被迅速冷冻。在进行电子显微镜处理后，这些相同的细胞将被ET分析，并在3D中对感兴趣的结构进行建模和分析。这种相关的光学和电子显微镜方法将是收集数据的一种极其有效的方法。本申请中建议的数据收集包括与健康直接相关的项目。我们将使用这些方法来表征用于研究乳腺癌的细胞培养模型系统的超微结构。我们将以高分辨率、三维细节分析结核分枝杆菌等细菌如何感染哺乳动物细胞，我们将研究几种细胞类型的细胞分裂，以了解特定分子如何影响这一过程。细胞分裂对癌症和异常发育研究的重要性是众所周知的。这些项目中的每一个都将极大地受益于能够拍摄对活细胞感兴趣的现象，并迅速将标本转移到HPF进行冷冻固定。自动断层扫描分析将极大地提高每个项目的数据吞吐量，使具有统计意义的样本大小成为可能。关联性。这项研究的目标是为完整细胞中的几个与疾病相关的过程提供高分辨率的结构背景，这些过程与分子遗传学和结构蛋白质组学提供的结构和功能信息相关。这项提案中要求的最先进的设备将大大有助于我们了解细菌感染、乳腺癌的机制，以及细胞如何分裂。反过来，这种对机制的更好理解将促进更有效的疾病干预治疗。

DESCRIPTION (provided by applicant): One of the most important developments in transmission electron microscopy in recent years has been instrumentation for automated electron tomography. Electron tomography (ET) produces 3D images of cell components at high resolution, helping bridge the gap between molecular structural biology and in vivo cell biological structure studies. In some tomography studies it is actually possible to recognize the signature structure of molecules within a 3-D model of the cell. Funds are requested for an Xplore 3D Retrofit package for a Tecnai 12 to upgrade an existing Tecnai 12 Biotwin electron microscope for automated tomographic data collection, and a Leica EMPACT2 high pressure freezer with Live Cell Imaging System to permit viewing of live cells in a light microscope, then to freeze them in about 5 seconds. This system of instruments will greatly enhance both the quality and quantity of electron tomographic data that can be obtained. Cells will be observed live in a light microscope and when they reach the right stage of the cell cycle or of development they will be rapidly frozen. After processing for electron microscopy, these same cells will be analyzed by ET and the structures of interest modeled and analyzed in 3D. This correlative light and electron microscopic approach will be an extremely efficient way to collect data. The data collection proposed in this application includes projects with direct health relevance. We will characterize the ultrastructure of a cell culture model system for studying breast cancer using these methods. We will analyze in high resolution, 3-dimensional detail how bacteria such as Mycobacterium tuberculosis infect mammalian cells, and we will study cell division in several cell types to understand how specific molecules can affect that process. The importance of cell division to research on cancer and abnormal development is well known. Each of these projects will benefit greatly by being able to film the phenomena of interest in live cells and rapidly transfer the specimen to the HPF for freeze fixation. Automated tomographic analysis will greatly improve the throughput of data for each project, making it possible to have statistically significant sample sizes. RELEVANCE. The goal of this research is to provide a high-resolution structural context for several disease-related processes in INTACT CELLS that correlates with the structural and functional information provided by molecular genetics and structural proteomics. The state-of-the-art equipment requested in this proposal will contribute significantly to our understanding of the mechanisms of bacterial infection, breast cancer, and how cells divide. In turn, this better understanding of mechanism will promote more effective treatments for disease intervention.