STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

这个项目的目标是表征形状，分子量 分布和单个大分子的元素组成， 大分子组装体 这种组装对于许多细胞来说是至关重要的。 功能，它们在体外的行为反映了它们的功能， 在完整的细胞中进行调节。 这个项目依赖于一种独特的仪器 - 低温高分辨率场发射扫描 透射电子显微镜（STEM）-用于分子量图谱 用平行电子能量损失谱进行化学分析 （皮）。 该仪器的研制，新的制备 直接冷冻薄膜和高分辨率铂的技术 复制品是去年描述的。 Now，the capabilities能力of this STEM干 已经扩展到直接冷冻组织的冷冻切片。 小脑皮质切片的质量映射和钙分析 特征性的两个不同类别的内质网（ER）在 浦肯野细胞树突。 此外，光谱成像，一个新的 PEELS用于高分辨率元素映射技术已经被 实现并用于成像钙螯合ER的分布 在这些树突中。 来测量水的分布 浦肯野细胞树突，一种基于低损耗分析的新方法 冷冻水合切片的低剂量PEELS图区域已被 开发 低剂量质量映射和高剂量PEELS的组合 光谱成像似乎是一种实用的关联方法 离子和水通量伴随神经元活动。

The goal of this project is to characterize the shape, molecular weight distribution, and elemental composition of individual macromolecules and macromolecular assemblies. Such assemblies are critical to many cell functions, and their behavior in vitro reflects their function and regulation in intact cells. This project depends on an unique instrument - a low-temperature, high-resolution, field-emission scanning transmission electron microscope (STEM) - for molecular weight mapping and chemical analysis by parallel electron energy loss spectroscopy (PEELS). The development of this instrument, and new preparation techniques for directly-frozen thin films and high-resolution platinum replicas were described last year. Now, the capabilities of this STEM have been extended to ultrathin cryosections of directly frozen tissues. Mass mapping and calcium analysis of sections of cerebellar cortex have characterized two distinct classes of endoplasmic reticulum (ER) in the dendrites of Purkinje cells. In addition, spectrum imaging, a new technique for high-resolution elemental mapping by PEELS, has bee,) implemented and used to image the distribution of calcium-sequestering ER within these dendrites. To measure the distribution of water within Purkinje cell dendrites, a new method based on analyzing the low-loss region of low-dose PEELS map of frozen hydrated sections has been developed. The combination of low-dose mass mapping and high-dose PEELS spectrum imaging would appear to be a practical method for correlating ion and water fluxes that accompany neuronal activity.