Improved data collection for FEI TF20 and Philips CM12 electron microscopes

说明(由申请人提供)：本建议书申请资金，用于为我们的FEI Tecnai F20(TF20)电子显微镜购买一台TVIPS 4K x 4K CCD摄像机，配备一个1K x 1K快速扫描CCD附件。TF20的主要用户的研究，包括六个由NIH资助的项目，提供了细菌发病机制、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领域的尖端和最先进的进展。小使用者进一步拓宽了我们的研究范围，由NIH资助的四项关于糖酵解途径、细菌中的氮代谢和线粒体呼吸途径的研究。为TF20购买新的CCD相机将使我们能够有效和准确地为以下主要项目收集数据：1)病原细菌黏附菌毛在人类宿主中特定器官定植的专门化比较研究，2)对凋亡体的研究，对线粒体凋亡途径至关重要的大分子组件的研究，3)关于“坏胆固醇”在心血管疾病中的作用的研究：低密度脂蛋白单独的结构和功能及其与其结合受体LDLR的复合体，4)蛋白质寡聚对脊髓灰质炎病毒RNA复制的作用的研究，包括通过蛋白质3AB分离和连接到膜上的3Dpol聚合酶的研究，5)在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复合体的比较研究，以及6)细丝调节骨骼肌收缩的研究。在我们现有的瞬变电磁基础上增加一个大面阵的电荷耦合器件，将使我们保持在各自领域的前沿。目前安装在TF20上的1K x 1K电荷耦合器件是用于像散校正、焦距调整和在胶片上记录低剂量图像后检查样品的极佳工具。所要求的大幅面电荷耦合器件捕捉的区域比我们当前的电荷耦合器件大十倍。这种样本成像数量的增加是至关重要的，因为我们目前的CCD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，现有的电荷耦合器件对于我们的飞利浦CM12电子显微镜的日常工作来说将是一项重要的资产，因此，这项建议的另一个关键好处将是将我们现有的TVIPS 1K x 1K电荷耦合器件转移到我们的飞利浦CM12电子显微镜，这不需要资金。CM12拥有庞大的用户基础，目前包括五个主要的TF20用户和七个额外的项目，其中六个是由NIH资助的。新用户也在稳步涌入。无论是作为培训工具还是CM12上的数据收集，在这台显微镜上安装一个电荷耦合器件都是非常有益的。公共卫生相关性：这项提案要求提供资金，将我们的FEI Tecnai F20电子显微镜升级为数字成像系统。电子显微镜用户的研究，包括美国国立卫生研究院资助的10个项目，在心血管疾病、细菌致病、细胞死亡(细胞凋亡)、病毒复制、肌肉调节、蛋白质生物合成和能源生成等领域提供了尖端的、最先进的进展。这种硬件的增加将显著提高所有这些研究项目的生产率，推动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.