3-Dimensional Fine Structure of Cells and Tissues

描述(由申请人提供)：这是一项更新博尔德实验室细胞三维电子显微镜研究资源资助的建议。我们的技术研究目标包括开发可靠的样品制备、图像采集和图像处理方法。我们设想的方法应该最终揭示细胞和细胞器在空间分辨率下的大体积三维结构，这是以前无法达到的。这一目标将通过玻璃化细胞的研究来实现，这种方法既保留了大规模的细胞组织，又保留了细胞内大分子复合体的原子细节。我们将致力于将细节可视化到2-3 nm，这一分辨率足以将大分子的原子模型对接到3-D冷冻电子显微镜图像中，方法是使用各种薄的冷冻水合样品的冷冻电子断层扫描。这些将包括非常薄的细胞、分离的细胞器和大分子复合体，以及通过冷冻显微切割法制备的切片。我们对大规模特征的研究将使用玻璃化样本，这些样本随后通过冷冻替代固定并嵌入塑料中进行显微切割，从而允许检查任何给定的哺乳动物细胞的大部分，无论是在培养中还是在其正常组织的背景下。这些方法将通过图像处理进行整合，以生成细胞结构和功能的全面视图。通过连接这些成像模式，我们应该能够解释来自光学显微镜和活细胞生理研究的数据，这些数据可以通过高分辨率冷冻断层扫描和原子分辨率结构相结合的方式观察到的分子细节。我们的技术研究在一定程度上是由我们与七个实验室的合作推动的，这些实验室的生物医学研究提出了一些科学问题，需要我们的方法应该提供的那种高分辨率和/或大容量的细胞三维信息。这些合作和我们的许多服务项目大多与人类疾病和障碍直接相关，因此我们的技术工作应该很快就会为医学提供信息。我们将研究的一些结构目前正在作为药物和其他治疗措施的靶点进行研究，因此我们详细的细胞和分子结构研究也应该有助于翻译研究。我们的提案还描述了培训和传播计划，因此我们的技术的使用将很快得到广泛应用。

DESCRIPTION (provided by applicant): This is a proposal to renew a Research Resource grant that supports the Boulder Laboratory for 3-D Electron Microscopy of Cells. Our goals for research in technology include the development of methods for reliable specimen preparation, image acquisition, and image processing. The methods we envision should ultimately reveal the 3-D structure of large volumes from cells and organelles at space resolutions that have previously been unattainable. This goal will be achieved through the study of vitrified cells, an approach that preserves both large-scale cellular organization and the atomic details of macromolecular complexes within cells. We will work to visualize details at 2 - 3 nm, a resolution sufficient to dock atomic models of macromolecules into 3-D cryo-EM images, by using cryo-electron tomography of various thin, frozen hydrated samples. These will including very thin cells, isolated organelles and macromolecular complexes, and sections prepared by cryo-microtomy. Our studies of large-scale features will use vitrified samples that are subsequently fixed by freeze-substitution and embedded in plastic for microtomy, allowing the examination of big fractions of any given mammalian cell, either in culture or within the context of its normal tissue. These approaches will be integrated through image processing to generate a comprehensive view of cellular structure and function. By connecting these imaging modalities, we should be able to interpret data from light microscopy and physiological studies of living cells in terms of the molecular details that can be observed through the combination of high-resolution cryo-tomography with atomic resolution structures. Our technological studies are motivated in part by our collaborations with seven labs whose biomedical research has posed scientific questions that require the kind of high resolution and/or large volume 3-D information about cells that our methods should provide. Most of these collaborations and many of our service projects are directly related to human diseases and disorders, so our technological work should soon inform medical science. Some of the structures we will study are currently under investigation as targets for drugs and other therapeutic measures, so our detailed cellular and molecular structure studies should also help translational research. Our proposal also describes plans for training and dissemination, so the use of our technologies will soon become widespread.