STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

这个项目的目标是描述形状， 分子量分布和元素组成 单个大分子和大分子组装体， 强调钙结合，和磷酸化，调节 proteins.本课题利用场致发射技术， 扫描透射电子显微镜（STEM） 能量损失谱（EELS）？一种新技术， 光谱成像？分析和绘制 生理上重要的元素，如P和Ca，分辨率为 优于10 nm。该技术的应用举例如下： 去极化时外周线粒体光谱成像 青蛙交感神经节的神经元。线粒体内Ca图 显示钙积累的焦点部位，小于10 nm， 直径;这些位点内的Ca浓度足够高 即使是单个像素的光谱也会显示出显著的Ca L23 EELS边缘。该信号的定量与 空间平均总Ca浓度约为10 mmol/kg干重。 这些结果首次表明， 分布图在一个单独的区域内。战略 单原子探测在高分辨率说明EELS 绘制一系列标本的图谱，包括烟草花叶病毒 (TMV)分离的DNA质粒和磷酸化蛋白质。 TMV的光谱成像产生对应于1-3 P的P图 在病毒颗粒的投影中心区域中每像素的原子数 在RNA链所在的区域，而在中心区域之外， P原子数为零。结果表明，单 磷原子在生物组件内是可检测的。

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.