Improved data collection for FEI TF20 and Philips CM12 electron microscopes

描述（由申请人提供）：本提案申请资助为我们的FEI Tecnai F20 （TF20）电子显微镜购买一台TVIPS 4K x 4K CCD相机，并附有1K x 1K Fastscan CCD附件。TF20主要用户的研究，包括6个nih资助的项目，在细菌发病机制、细胞凋亡、心血管疾病、病毒复制、蛋白质生物发生和肌肉调节等领域提供了最前沿的、最先进的研究进展。次要用户进一步拓宽了我们的研究与四个美国国立卫生研究院资助的研究糖酵解途径，细菌氮代谢和线粒体呼吸途径。为TF20购买新的CCD相机将使我们能够高效准确地收集以下主要项目的数据：1)病原细菌粘附菌毛在人类宿主特定器官定定的特化比较研究，2)凋亡研究，线粒体凋亡途径的关键大分子组装，3)研究“坏胆固醇”在心血管疾病中的作用。LDL低密度脂蛋白及其结合受体（LDLR）的结构和功能；4)蛋白质寡聚化在脊髓灰质炎病毒RNA复制中的作用研究，包括分离的3Dpol聚合酶和通过蛋白3AB粘附在膜上的研究；5)哺乳动物和细菌中介导新生蛋白共翻译易位的核糖体- sec61和核糖体- secy复合物的比较研究；6)细丝对骨骼肌收缩调控的研究。在我们现有的TEM上增加一个大幅面CCD将使我们保持在各自领域的前沿。目前在tf20上的1K x 1K CCD是一个很好的工具，用于散光校正，对焦调整，以及在胶片上记录低剂量图像后检查样品。所要求的大画幅CCD捕获的图像比我们现有的CCD大十倍。样品成像量的增加是必不可少的，因为我们目前的CCD所施加的限制排除了收集高分辨率图像重建所需的大型数据集的可能性。然而，现有的CCD对于我们的飞利浦CM12电子显微镜的日常工作将是一个重要的资产，因此，本提案的另一个关键好处是将我们现有的TVIPS 1K x 1K CCD转移到我们的飞利浦CM12电子显微镜上，而不需要资金。CM12拥有庞大的用户群，目前包括5个主要的TF20用户和7个额外的项目，其中6个是美国国立卫生研究院资助的。新用户也在不断涌入。在这台显微镜上安装CCD将是非常有益的，无论是作为培训工具，还是在CM12上收集数据。与公共卫生相关：本提案申请资金，以升级我们的FEI Tecnai F20电子显微镜与数字成像系统。电子显微镜用户的研究，包括10个美国国立卫生研究院资助的项目，在心血管疾病、细菌发病机制、细胞死亡（凋亡）、病毒复制、肌肉调节、蛋白质生物合成和能量产生等领域提供了最前沿的、最先进的进展。这些硬件的增加将显著提高所有这些研究项目的生产率，推进预防或消除人类疾病的研究。

DESCRIPTION (provided by applicant): This proposal requests funding for the purchase of a TVIPS 4K x 4K CCD camera for our FEI Tecnai F20 (TF20) electron microscope, with a 1K x 1K Fastscan CCD accessory. Research by the major users of the TF20, including six NIH-funded projects, provide cutting edge, state-of-the-art advances in the fields of bacterial pathogenesis, apoptosis, cardiovascular disease, viral replication, protein biogenesis, and muscle regulation. Minor users broaden our research further with four NIH-funded studies on the glycolytic pathway, nitrogen metabolism in bacteria, and the mitochondrial respiratory pathway. The purchase of a new CCD camera for the TF20 will allow us to collect data efficiently and accurately for the following major projects: 1) comparative studies on the specialization of pathogenic bacterial adhesion pili for colonization of specific organs in their human hosts, 2) studies on the apoptosome, a macromolecular assembly that is critical to the mitochondrial apoptosis pathway, 3) studies on the role of `bad cholesterol' in cardiovascular disease: the structure and function of LDL low density lipoproteins alone and in complex with its binding receptor, LDLR, 4) studies on the role of protein oligomerization for the replication of poliovirus RNA, including studies on the 3Dpol polymerase in isolation and tethered to membranes via the protein 3AB, 5) comparative studies of ribosome-Sec61 and ribosome-SecY complexes that mediate co-translational translocation of nascent proteins in mammals and bacteria, and 6) studies on the regulation of skeletal muscle contraction by thin filaments. The addition of a large format CCD to our existing TEM will allow us to remain at the forefront of our respective fields. The 1K x 1K CCD that is currently on theTF20 is an excellent tool for astigmatism correction, focus adjustment, and for checking a sample after a low dose image has been recorded on film. The requested large format CCD captures images of an area that is ten times larger than our current CCD. This increase in the amount of sample imaged is essential, as the limits imposed by our current CCD exclude the possibility of collecting the large datasets required for high resolution image reconstructions. However, the existing CCD will be a significant asset for routine work that is done on our Philips CM12 electron microscope Thus, an additional key benefit of this proposal will be the transfer of our existing TVIPS 1K x 1K CCD to our Philips CM12 electron microscope, for which no funding is requested. The CM12 has a large user base that currently includes the five major TF20 users and seven additional projects, six of which are NIH-funded. There is also a steady influx of new users. Having a CCD on this microscope will be extremely beneficial both as a training tool and for data collection on the CM12. Public Health Relevance: This proposal requests funding to upgrade our FEI Tecnai F20 electron microscope with a digital imaging system. Research by users of the electron microscope, including 10 NIH-funded projects, provide cutting edge, state-of-the-art advances in the fields of cardiovascular disease, bacterial pathogenesis, cell death (apoptosis), viral replication, muscle regulation, protein biosynthesis, and energy generation. Addition of this hardware will significantly improve the productivity of all these research projects, advancing research toward the prevention or elimination of human disease.