3 DIMENSIONAL FINE STRUCTURE OF CELLS AND TISSUES

我们寻求对技术开发的支持，以帮助 阐明细胞和组织的三维精细结构。 将采用两种成像方法：(1)厚样品 生物材料将在JEM-1000高压电子设备中进行研究 显微镜(HVEM)和系列倾斜视图将被成像以提供 有关标本的三维结构的信息；(2) 不同厚度的图像将在HVEM中或通过常规成像 并通过电子显微镜和组装成三维模型的组合图像 处理和计算机图形学。我们努力的主旨将是 为这些成像方法开发样品制备技术。 (一)继续开展结构神经生物学研究，完善 现有的特定细胞的选择性染色方法和 分子。层析成像方法将与立体建模和 连续切片重建以提取有关的定量信息 神经元几何学。(2)我们将调查高压的效用 用于研究太大而不能很好保存的标本的快速冷冻 传统的冷冻方法。然后，冷冻固定材料将被 设计了几种辅助方法，以允许可靠地可视化细微 结构和特定抗原的位置。(三)我们将探索 冷冻超高速电子显微镜在冷冻水化材料研究中的应用 鞭毛和基体作为测试系统。(4)我们会继续 EM水平上原位杂交方法的研究进展 用生物素标记的探针定位核酸。

We seek support for the development of technologies that will help elucidate the three-dimensional (3-D) fine structure of cells and tissues. Two approaches to imaging will be employed: (1) thick samples of biological material will be studied in a JEM-1000 high voltage electron microscope (HVEM), and serial tilted views will be imaged to provide information about the 3-D structure of the specimen; (2) serial sections of various thickness will be imaged either in the HVEM or by conventional electron microscopy and assembled into 3-D models by a combination of image processing and computer graphics. The major thrust of our efforts will be to develop specimen preparation technologies for these imaging approaches. (1) We will continue our work on structural neurobiology, improving existing methods for the selective staining of specific cells and molecules. Tomographic methods will be combined with stereo modeling and serial section reconstruction to extract quantitative information about neuronal geometry. (2) We will investigate the utility of high pressure rapid freezing for the study of specimens too large to be well preserved by conventional freezing methods. Cryofixed material will then be treated by several secondary methods designed to permit reliable visualization of fine structure and of the location of specific antigens. (3) We will explore the utility of cryoHVEM for the study of frozen hydrated material, using flagella and basal bodies as test systems. (4) We will continue our development of methods for in situ hybridization at the EM level to reveal the location of nucleic acids with biotinylated probes.