This is a request for funds to replace a 25 year old, heavily used Balzers 360M freeze-etch apparatus with a state-of -the-art BAL-TEC 060 instrument that will be housed in the cryo-techniques laboratory of the Electron Microscope Service of the Department of Molecular, Cellular and Developmental Biology. During its life-tide the old Balzers 360M has produced micrographs used in over 120 research papers. The most important reasons for requesting funds for a new instrument are the renewed interest of the PI in freeze-fracture /-etch electron microscopy based on experiences gained during his 1992 sabbatical leave in Zurich, Switzerland, and the recognition that his research and that of several other members of the department could profit significantly from having access to a state-of-the-art freeze-fracture /-etch machine. In addition, the Balzers 360M apparatus is simply worn out after over 15,000 runs as evidenced by the frequent breakdowns and relatively poor vacuum performance (4x10-6 Torr during routine operation versus about 10-8 Torr for a new cryo-pumped BAL-TEC 060 instrument) despite frequent maintenance. Finally, BAL-TEC Inc. has informed us that they will no longer stock spare parts for the Balzers 360M after the end of 1993. The daily operation of the new instrument will be supervised by the Director of the EM Service, Dr. Thomas Giddings, an internationally known expert in freeze-fracture /-etch electron microscopy. The initial projects that will make use of the new instrument include 1) the analysis of unique, transient plasma membrane configurations associated with secretion and membrane recycling in turgid, walled cells; 2) the elucidation of the spatial organization of the three proteins of the oxygen-evolving complex on the surface of photosynthetic membranes; 3) the characterization of subdomains of cytoplasmic dynein complexes; 4) the elucidation of the surface topography of crystallized photosystem II complexes; and 5) the analysis of native membrane protein complexes directly eluted from bands of nondenaturing agarose gels.
