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器透射电子显微镜(EFTEM)。这些仪器对于丹福斯中心将完成的最先进的电子显微镜来说将是必不可少的。Balzers高压冷冻机允许通过物理过程保存用于电子显微镜的植物细胞：对植物组织进行超快速冷冻。与化学固定相比，高压冷冻固定的细胞表现出良好的细胞质、细胞器和微管、分泌结构等微细结构的保存，并具有最小的收缩和肿胀伪影。当用于免疫标记目的时，这些细胞也具有高度的抗原性。这种物理固定过程(冷冻固定)是目前最实用的方法，可以均匀固定从小溶质到大分子络合物的细胞成分。这些品质改善了对细胞的原位分子环境的分析，对于实现我们在丹福斯中心细胞生物学研究的目标至关重要。冷冻固定和冷冻替代样品的薄片将在配备有能量过滤器和CCD摄像机的电子傅立叶变换电子显微镜中成像。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。与传统的透射电子显微镜相比，这在产生样品对比度方面提供了更大的灵活性。常规薄片可以在高对比度下成像，即使没有切片染色或切片染色较差，而为优化抗原性而准备的免疫金标记切片(但通常对比度较差)可以调整为最佳对比度，而不会对重金属染色。能量滤光片可以用来最大限度地减少色差，使之能够清晰地成像厚切片。与EFTEM的平台倾斜式测角仪相结合，可以通过电子断层扫描对细胞进行深度分析。EFTEM可以用来在薄片上绘制元素分布图，并结合冷冻固定和冷冻替代，这为分析植物组织中可溶性元素的分布提供了可能性。目前丹佛斯公司将使用该设备的项目包括：在植物细胞感染过程中，用厚层电子断层扫描、薄层电子显微镜以及病毒蛋白的免疫金定位来研究植物致病病毒(烟草病毒和双生病毒)的细胞和分子生物学。所有样本都将进行高压冷冻和冷冻替代，以最好地保存感染事件。其他项目包括研究Rab蛋白介导的植物细胞膜运输；参与肌苷一磷酸代谢的酶复合体和木质素生物合成酶的免疫金定位；植物疫苗产生的抗原的免疫定位以研究它们在哺乳动物组织中的靶向；以及使用深度蚀刻旋转阴影方法研究植物组织中的胞间连丝结构。我们还鼓励区域研究所的科学家使用这些仪器。这一仪器将是推进丹福斯中心使命的关键要素。丹福斯中心致力于应用基础植物生物学的新知识，以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学生、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.