The purpose of this grant request is to obtain funds to purchase and install a High Pressure Freezer (HPF) machine. This unit will be used to prepare well-preserved specimens for tomography applications. High pressure freezing delivers the best specimen preparation method for preserving cell and tissue ultrastructure and therefore, obtaining high resolution in situ data. The requested equipment is a Bal-Tec 11PM 010 High Pressure Freezing Machine (HFP) and accessories for sample preparation. Our research programs in structural biology involve the three-dimensional reconstruction of cells and tissues using thick sections and intermediate voltage electron microscopy (IVEM). We coordinate and integrate our structural studies with data obtained from molecular microscopy, X-ray crystallography, biochemical, biophysical and neurophysiological techniques. Our goal is to accurately reconstruct the structure of cells and organelles using the best preparative, microscopy and computational methods. At present, the closest HFP units are located in the San Francisco Bay Area or the Tucson, Arizona. The HFP will be housed in the National Center for Microscopy and Imaging Research at the University of California, San Diego. The five major programs requiring the use of the HFP are: (1) Three-dimensional cellular organization of gap junction membrane channels (Sosinsky, P.!.); (2) Structural studies of cellular components in the Node of Ranvier (Ellisman, P.1.); (3) Changes in macromolecular associations in cerebellar Purkinje neuron dendritic spines during synaptic-depression (Tsien and Lev-Ram, P.1?s); (4) Mechanisms of non-enveloped virus entry (Johnson and Schneemann, P.I.?s); and (5) Three-dimensional structure and function of mitochondria as viewed by electron tomography (Frey, P.1.). In addition, four collaborative projects from the NCMIR Research Resource that would immediately benefit from the usage of the HFP are described briefly. These nine research projects involve over 25 investigators that would depend heavily on the use of the HPF. In all of these research projects, the primary goal is to understand the three-dimensional structure of organized cellular components and relate this morphology to their functionality.
| Sosinsky, Gina E; Crum, John; Jones, Ying Z et al. (2008) The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications. J Struct Biol 161:359-71 |
