One of the important advances in transmission electron microscopy in recent years has been development of methods for observing samples embedded in non-crystalline (vitreous) ice. These samples are about as near to the native site as possible for observation by electron microscopy. Funds are requested for a Phillips CM120 Transmission EM equipped for cryoEM operation so that this technology can be readily available to researchers at the University of California, Berkeley and the San Francisco Bay Area. We propose to use cryoEM to study vesicle fractions from the S. cerevisiae secretory pathway, liposomes, beam- sensitive artificial polymers used in bone-replacement surgery, Mycobacterium tuberculosis, nano-crystals, and a number of different 2-D protein crystals. In addition, we have a number of core users who will use the microscope in conventional mode, but who will benefit from having a modern microscope with improved resolution and higher accelerating voltage. This group is primarily interested in using the microscope for EM immunolocalization work and/or characterizing mutant phenotypes in teleost fish retina, mammalian stomach cells, S. cerevisiae and S. pombe yeasts, Drosophila and C. elegans. The lower material will be prepared by high pressure freezing and freeze substitution for optimum preservation of morphology and antigenicity. The microscope will be housed in the Electron Microscope Laboratory at the University of California, Berkeley, a fee-for-service facility open to all campus users as well as researchers from the surrounding institutions such as UC San Francisco, UC Davis, UC Santa Cruz and Stanford. As far as we known, none of these schools has a cryoEM available for general use. All of the major uses in this proposal are experienced EM users and most have NIH funding. Selected core users plus the Supervisor of the EM Lab will make up an advisory committee to oversee use of the instrument and set policy. Use by other regional investigators will be encouraged in the time remaining after core investigator use. UC Berkeley will provide space and staff for operating the microscope as well as $40,000 for an extended service contract.
