Acquisition of a Transmission Electron Microscope

本提案要求拨款支持购置一台日立H-7100透射电子显微镜（TEM）和辅助设备。这项文书对该部的研究活动至关重要，并将在各级教育和人力资源开发方面发挥重要作用。日立H-7100仪器在未来可能的升级方面具有优异的性能特点和灵活性。我们建议为这个仪器配备数字图像的收集和存储。我们将能够利用光学显微镜成像和生物技术中心（美国国家科学基金会科学技术中心）和匹兹堡超级计算中心现有的专业知识和计算资源，协助处理、分析和数字图像的三维重建。这两个中心都位于梅隆学院，那里有生物科学系和电子显微镜设施。生物科学系现有的唯一仪器是飞利浦300透射电镜，已有25年以上的历史。它的能力非常有限，因为很难或不可能配备几种现代外围组件和附件，包括视频/数字成像设备，测角仪或冷却级，以及STEM-SEM或EDS系统。此外，这种模式的年龄引起了对仪器部件甚至服务合同的持续可用性的担忧。因此，一台具有扩展能力的新显微镜对于支持生物科学系当前和未来的研究活动至关重要。在该系的18名教员中，7名将是该仪器的主要使用者，预计至少还有5名是次要使用者。这12位教职员工，占生物科学系教职员工的三分之二，在他们的研究项目中使用电子光学方法作为基本工具。所要求的仪器将用于旨在了解基本细胞、发育、神经生物学和生理过程的研究项目。主要用户项目将包括：1)旨在了解培养和活胚胎中细胞运动的相关电子显微镜和光光学研究（Ettensohn, Pollock, Taylor）；2)高分辨率免疫定位研究，重点关注在胚胎发育（Ettensohn, Pollock）和关键细胞生物学过程(如蛋白质靶向（Jones），运动性（Jarvik, Taylor）和能量代谢（Koretsky）中具有重要功能的蛋白质；3)突变体表型的超微结构分析；例如，细胞器结构或功能改变的细胞（Jarvik, Jones, Koretsky），果蝇正常和突变的视觉系统（Pollock），以及酶同型分子遗传转换后转基因小鼠的组织（Koretsky）；4)电镜原位杂交测定mrna的亚细胞分布；(波洛克);5)运动蛋白激酶的结构研究，包括在不同离子条件下蛋白质的构象变化和激酶-微管相互作用（Hackney）。该工具还将对本科、研究生和博士后层次的教育和人力资源开发产生重大影响。该系60名研究生、165名生物学专业本科生和20名博士后中，有相当一部分人将通过现有的正式电子光学方法课程（电子显微镜技术，BSC 03~20）和/或在他们的独立研究项目中使用所要求的仪器。3

This proposal requests funds to support the acquisition of a Hitachi H-7100 transmission electron microscope (TEM) and ancillary equipment. This instrument will be critical for the research activities of the Department and will play an essential role in education and human resources development at all levels. The Hitachi H-7100 instrument has excellent performance characteristics and flexibility in terms of possible future upgrading. We propose to equip this instrument for the collection and storage of digital images. We will be able to take advantage of existing expertise and computational resources in the Center for Light Microscope Imaging and Biotechnology (a N.S.F. Science and Technology Center) and the Pittsburgh Supercomputing Center to assist in the processing, analysis, and 3-D reconstruction of digital images. Both Centers are located in the Mellon Institute, where the Department of Biological Sciences and its Electron Microscope Facility are located. The single existing instrument in the Department of Biological Sciences, a Philips 300 TEM, is more than 25 years old. It is extremely limited in its capabilities because it is difficult or impossible to equip with several kinds of modern peripheral components and accessories, including video/digital imaging devices, goniometer or cooled stages, and STEM-SEM or EDS systems. Moreover, the age of this model raises concerns about the continued availability of parts and even service contracts on the instrument. A new microscope with expanded capabilities is therefore essential for the support of both current and future research activities in the Department of Biological Sciences. Of 18 faculty members in the Department, 7 will be major users of the instrument, and at least an additional 5 are anticipated to be minor users. All 12 of these faculty members, representing two-thirds of the faculty of the Department of Biological Sciences, use electron optical methods as an essential tool in their research programs. The requested instrument will be used in research projects aimed at an understanding of fundamental cell, developmental, neurobiological, and physiological processes. Major-user projects will include: 1) Correlated electron microscopic and light optical studies aimed at understanding cell movements in culture and in living embryos (Ettensohn, Pollock, Taylor); 2) Highresolution immunolocalization studies, focusing on proteins with important functions in embryonic development (Ettensohn, Pollock) and in critical cell biological processes such as protein targeting (Jones), motility (Jarvik, Taylor), and energy metabolism (Koretsky); 3) Ultrastructural analysis of mutant phenotypes; e.g., of cells with altered organelle structure or function (Jarvik, Jones, Koretsky), of normal and mutant visual systems in Drosophila (Pollock), and of tissues in transgenic mice following molecular genetic switching of enzyme isotypes (Koretsky); 4) Determination of the subcellular distribution of mRNAs by electron microscopic in situ hybridization; (Pollock); and 5) Structural studies of the motor protein kinesin, including conformational changes in the protein under varying ionic conditions and kinesin-microtubule interactions (Hackney). The instrument will also have a major impact on education and human resources development at the undergraduate, graduate, and postdoctoral levels. A significant number of the Department's 60 graduate students, 165 undergraduate biology majors, and 20 postdoctoral fellows will be expected to use the requested instrumentation either through an existing, formal course in electron optical methods (Techniques in Electron Microscopy, BSC 03~20) and/or in their independent research projects. 3