Acquisition Of A High Pressure Freezer And EFTEM

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A grant has been awarded to Dr. R. Howard Berg at the Donald Danforth Plant Science Center, St. Louis, Missouri, to acquire a high pressure freezer, a freeze substitution unit, and an energy filter transmission electron microscope (EFTEM). These instruments will be essential for the state of the art electron microscopy that will be done at the Danforth Center. The Balzers High Pressure Freezer permits preservation of plant cells for electron microscopy by a physical process: ultra-rapid freezing of plant tissues. Compared to chemical fixation, cells fixed by high pressure freezing show excellent preservation of cytoplasm, organelles, and delicate structures such as secretory structures and microtubules, and with minimum shrinkage and swelling artifacts. These cells also have heightened antigenicity when used for immunolabeling purposes. This physical fixation process (cryofixation) is currently the most practical method for uniform immobilization of cell components from small solutes to large macromolecular complexes. These qualities improve analysis of the in situ molecular environment of cells and are essential for meeting our goals in cell biology studies at the Danforth Center. Thin sections of cryofixed and freeze-substituted specimens will be imaged in an EFTEM, equipped with an energy filter and CCD camera. The energy filter is an in-column electron spectrometer that is used to select electrons of the appropriate energies (from those scattered by the specimen) to generate specimen contrast. Compared to a conventional TEM, this gives much more flexibility in generating specimen contrast. Conventional thin sections can be imaged in high contrast, even with no or poor section staining, and immunogold-labeled sections prepared to optimize antigenicity (but normally poor in contrast) can be adjusted to optimal contrast without staining in heavy metals. The energy filter can be used to minimize chromatic aberration, making it possible to image thick sections with clarity. Coupled with the EFTEM's stage-tilting goniometer, this permits depth analysis in cells via electron tomography. The EFTEM can be used to map elemental distribution in thin sections and, coupled with cryofixation and freeze substitution, this gives the potential to analyze the distribution of soluble elements in plant tissues. The current projects from Danforth personnel that will use this equipment include: investigation of the cell and molecular biology of plant pathogenic viruses (tobamoviruses and geminiviruses) by thick section electron tomography, thin section TEM, and immunogold localization of viral proteins, during infection of plant cells. All samples will be high pressure-frozen and freeze substituted, to optimally preserve infection events. Other projects include investigation of Rab protein-mediated membrane trafficking in plant cells; immunogold localization of enzyme complexes involved in inosine monophosphate metabolism, and of enzymes of lignin biosynthesis; immunolocalization of antigens produced by plant vaccines to investigate their targeting in mammalian tissue; and use of the deep etch rotary shadowing method to investigate plasmodesma structure in plant tissues. We are also encouraging scientists from regional institutes to make use of these instruments. This instrumentation will be a key element in furthering the mission of the Danforth Center. The Danforth Center is dedicated to applying new knowledge in basic plant biology to help sustain productivity in agriculture, and to contribute to the education and training of graduate and postdoctoral students, scientists and technicians from around the world.