3-DIMENSIONAL FINE STRUCTURE OF CELLS AND TISSUES

我们寻求支持，以开发有助于 阐明细胞和组织的三维（3-D）精细结构。 将采用两种成像方法：（1）厚样本 生物材料将在JEM-1000高压电子 显微镜（HVEM），和系列倾斜的意见将成像，以提供 关于标本的三维结构的信息;（2）连续切片 各种厚度将在HVEM中或通过常规的 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。 我们努力的主要方向将是 为这些成像方法开发样品制备技术。 (1)我们将继续在结构神经生物学方面的工作， 用于选择性染色特定细胞的现有方法， 分子。 层析成像方法将与立体建模相结合， 连续切片重建，以提取有关 神经元几何学 (2)我们将研究高压的效用 用于研究因太大而无法保存完好的标本的快速冷冻， 传统的冷冻方法。 冷冻固定的材料将通过 设计了几种辅助方法，以允许精细的可靠可视化 结构和特定抗原的位置。 (3)我们将探讨 cryoHVEM用于冷冻水合材料研究的实用性，使用 鞭毛和基体作为测试系统。 (4)我们将继续 在EM水平上开发原位杂交方法， 用生物素化探针定位核酸。

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.