STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

这个项目的目标是描述形状的特征， 聚合物的分子量分布和元素组成 单个大分子和大分子组装体，具有 强调钙结合和调节蛋白的磷酸化 蛋白质。该项目利用了场发射技术的优势。 带电子扫描电子显微镜(STEM) 能量损失谱(EELS)？一种名为 光谱成像？分析并绘制…的分布图 生理上重要的元素，如磷和钙，分辨率为 优于10 nm。这项技术的应用如下所示 去极化状态下外周线粒体的光谱成像 蛙交感神经节神经元。线粒体内钙图谱 显示钙积聚的焦点位置小于10 nm 直径；这些部位内的钙浓度足够高 即使是单个像素的光谱也显示出显著的情况 123号鳗鱼边。这一信号的量化与 空间平均总钙浓度为~10 mmol/kg干重。 这些结果首次显示了一个定量的元素 单个线粒体内的分布图。战略以实现 用EELS说明了高分辨率的单原子探测 包括烟草花叶病毒在内的一系列标本的作图 (TMV)、分离的DNA质粒和磷酸化蛋白。 TMV的光谱成像产生了对应于1-3P的P图 病毒粒子投射中心区域的每像素原子 RNA链所在的位置，而在中心区之外 P原子的数目为零。结果表明，单一的 磷原子在生物组装中是可以检测到的。

The goal of this project is to characterize the shape, molecular weight distribution and elemental composition of individual macromolecules and macromolecular assemblies, with emphasis on calcium binding by, and phosphorylation of, regulatory proteins. This project takes advantage of combining field-emission scanning transmission electron microscopy (STEM) with electron energy loss spectroscopy (EELS) ? a new technique known as spectrum imaging ? to analyze and map the distribution of physiologically important elements like P and Ca at a resolution of better than 10 nm. Application of this technology is illustrated by spectrum imaging of peripheral mitochondria in depolarized neurons of frog sympathetic ganglia. Intramitochondrial Ca maps reveal focal sites of Ca accumulation that are less than 10 nm in diameter; the Ca concentration within these sites is sufficiently high that a spectrum from even a single pixel exhibits a prominent Ca L23 EELS edge. Quantitation of this signal is consistent with a spatially averaged total Ca concentration of ~10 mmol/kg dry wt. These results show for the first time a quantitative elemental distribution map within an individual mitochondrion. Strategies for single atom detection at high resolution are illustrated by EELS mapping of a series of specimens including tobacco mosaic virus (TMV), isolated DNA plasmids, and phosphorylated proteins. Spectrum imaging of TMV yields P maps that correspond to 1-3 P atoms per pixel in the projected central region of the viral particles where the RNA strand lies, whereas outside the central region the number of P atoms is zero. The results demonstrate that single atoms of phosphorus are detectable within biological assemblies.