Acquisition Of A High Pressure Freezer And EFTEM

密苏里州圣路易斯市唐纳德丹福斯植物科学中心的R. Howard Berg博士获得了一笔赠款，用于购买一台高压冷冻机、一台冷冻替代装置和一台能量过滤透射电子显微镜（EFTEM）。这些仪器将是丹佛斯中心最先进的电子显微镜所必需的。巴尔查斯高压冷冻机允许保存植物细胞的电子显微镜通过物理过程：植物组织的超高速冷冻。与化学固定相比，高压冷冻固定的细胞可以很好地保存细胞质、细胞器和精细结构，如分泌结构和微管，并且最小的收缩和肿胀。当用于免疫标记时，这些细胞也具有较高的抗原性。这种物理固定过程（冷冻固定）是目前将细胞成分从小溶质均匀固定到大分子复合物最实用的方法。这些特性提高了对细胞原位分子环境的分析，对于实现我们在丹佛斯中心细胞生物学研究的目标至关重要。冷冻和冷冻替代标本的薄片将在配备能量过滤器和CCD相机的EFTEM中成像。能量过滤器是一种柱内电子能谱仪，用于选择适当能量的电子（从样品散射的电子中）以产生样品对比度。与传统的透射电镜相比，这在生成标本对比度方面具有更大的灵活性。传统的薄片可以在高对比度下成像，即使没有或较差的切片染色，而制备的免疫金标记的切片可以优化抗原性（但通常对比度较差），可以调整到最佳对比度，而无需在重金属中染色。能量滤光片可用于最小化色差，使其有可能成像厚切片与清晰度。结合EFTEM的阶段倾斜角计，这允许通过电子断层扫描对细胞进行深度分析。EFTEM可以用来绘制元素在薄片上的分布，再加上冷冻固定和冷冻取代，这就有可能分析植物组织中可溶性元素的分布。丹佛斯人员目前将使用该设备的项目包括：在植物细胞感染期间，通过厚层电子断层扫描、薄层透射电镜和病毒蛋白的免疫金定位来研究植物病原病毒（托巴莫病毒和双病毒）的细胞和分子生物学。所有样品将被高压冷冻和冷冻替代，以最佳地保存感染事件。其他项目包括研究Rab蛋白介导的植物细胞膜运输；肌苷单磷酸代谢酶复合物和木质素生物合成酶的免疫金定位植物疫苗产生抗原的免疫定位研究其在哺乳动物组织中的靶向性并利用深蚀刻旋转阴影法对植物组织内的胞浆连丝结构进行了研究。我们还鼓励区域研究所的科学家利用这些仪器。这种仪器将是推进丹佛斯中心使命的关键因素。丹佛斯中心致力于应用基础植物生物学的新知识来帮助维持农业生产力，并为来自世界各地的研究生和博士后、科学家和技术人员的教育和培训做出贡献。

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.