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
