Acquisition of a Transmission Electron Microscope

该提案要求提供资金，以支持采购日立H-7100透射式电子显微镜(TEM)和辅助设备。这项文书将对新闻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出色的性能特征和灵活性。我们建议为这台仪器配备收集和存储数字图像的设备。我们将能够利用光学显微镜成像和生物技术中心(NS.F.科学和技术中心)和匹兹堡超级计算中心的现有专业知识和计算资源，帮助处理、分析和三维重建数字图像。这两个中心都位于梅隆研究所，生物科学系及其电子显微镜设施就设在那里。生物科学系现有的唯一仪器--飞利浦300电子显微镜--已经有25年以上的历史了。它的能力极其有限，因为它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、测角仪或冷却工作台，以及STEM-SEM或EDS系统。此外，这种模式的出现引发了人们对该仪器上部件甚至服务合同的持续供应的担忧。因此，具有扩展能力的新显微镜对于支持生物科学系目前和未来的研究活动是必不可少的。在该系的18名教职员工中，有7人将是该仪器的主要使用者，预计至少还有另外5人将是次要使用者。所有这12名教职员工，代表了生物科学系三分之二的教职员工，在他们的研究项目中使用电子光学方法作为必要的工具。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学研究(Ettensohn，Pollock，Taylor)；2)高分辨率免疫定位研究，侧重于在胚胎发育(Ettensohn，Pollock)和关键细胞生物学过程中具有重要功能的蛋白质，如蛋白质靶向(Jones)、运动性(Jarvik，Taylor)和能量代谢(Koretsky)；3)突变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(Pollock)正常和突变视觉系统的研究，以及酶同型转换后转基因小鼠组织的研究(Koretsky)，4)通过电子显微镜原位杂交确定mRNAs的亚细胞分布，(Pollock)，以及5)运动蛋白kinesin的结构研究，包括在不同离子条件下蛋白质的构象变化和运动微管与微管的相互作用(Hackney)。该工具还将对本科生、研究生和博士后层面的教育和人力资源开发产生重大影响。该系60名研究生、165名生物专业本科生和20名博士后研究员中的相当一部分人将通过现有的电子光学方法(电子显微镜技术，BSC 03~20)正规课程和/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