In January 2003, the journal Science listed Cryo Electron Tomography (GET) as one of the 10 Scientific Breakthroughs of 2002. Thanks to improvements in specimen preparation, EM instrumentation, software and hardware it is now more realistically possible to collect high-resolution tomographic data from samples embedded in vitreous ice. There are, or will be, electron microscopes at DC Berkeley, DC San Francisco, UC Davis, and Stanford to perform GET. Work on small, whole cells is an exciting, though limited option for many biologists. Working with vitreous cryosections of larger cells and tissues offers more possibilities for most researchers. To produce vitreous cryosections for GET, funds are requested for a Leica EM UC6 cryo-ultramicrotome and essential accessories. In addition to the cutting edge application of GET, this equipment will allow us to cut Tokuyasu-type cryosections for EM immunolabeling, and ultrathin vitreous cryosections for conventional, i.e., non-tomographic cryo-imaging. One or more of all these approaches will be used on wild type and mutant phenotypes in C. elegans, S. cerevisiae, Mycobacterium tuberculosis, mammalian gastric glands, inner ear hair cell stereocilia and kidney podocytes. All material for vitreous cryosections will be prepared by high pressure freezing. The microtome will be housed in the Electron Microscopy Laboratory at the University of California, Berkeley, a fee-for-service core facility open to all campus users as well as researchers from surrounding institutions such as UC Davis, UC Santa Cruz and Stanford. None of these institutions yet has the combination of high-pressure freezer and cryo-microtome required for vitreous cryo-sectioning. All the major users in this proposal are experienced EM users and most have NIH funding. Selected core users plus the Director of the EM Lab will make up an Advisory Committee to oversee use of the instrument and set policy. Use by regional investigators will be encouraged in the time remaining after core investigator use. UC Berkeley will provide space, staff, and funds for maintaining the equipment.
