MASS MEASUREMENT OF SINGLE MOLECULES WITH THE STEM

使用 STEM 对单分子进行质量测量

• 批准号: 3103995
• 负责人: JOSEPH S WALL
• 金额: $ 43.37万
• 依托单位: ASSOCIATED UNIV-BROOKHAVEN NATL LAB
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3103995
• 关键词: biomedical equipment resource; electron microscopy; image processing; molecular weight; scanning electron microscopy

The scanning transmission electron microscope (STEM) can provide low dose, high resolution images of unstained biological samples. Isolated objects or discrete image features can be integrated (after subtraction of substrate) to determine molecular weights and so provide a direct link between biochemistry and electron microscopy. The accuracy of STEM melecular weights improves from about 20% at 10 kD to about 4% at 100 kD and better than 1% above one million D for single particles imaged at a dose of 10 el/Angstroms squared. Practical results on tRNA, nucleosomes, fibrinogen, RNA polymerase, glutamine synthetase, 30s and 50s ribosomal subunits, filamentous viruses and TMV are in good agreement with theoretical predictions. Standard error of the mean can be reduced by averaging over 100-1,000 particles. Histograms of particle mass values can be used to characterize the homogeneity of the preparation. This technique has been applied routinely to a wide variety of users'/collaborators' samples over the past years at the Brookhaven STEM Biotechnology Resource. We propose to continue these productive collaborations and service activities while refining practical aspects of the technique. Further reduction of specimen temperature to 20 K is expected to reduce mass loss below 0.25%/el Angstroms squared now found at 120 K (8x less than at 300 K), permitting higher dose and higher accuracy. Reduction of surface migration at lower temperature is expected to lead to less redistribution of mass during radiation damage. This prediction will be tested using control specimens of known structure (e.g. TMV). A major new thrust will involve use of image averaging programs to improve signal to noise ratio in low dose images. This should permit extension of domainal mass mapping already demonstrated on fibrinogen and dynein to allow detection of the binding of small molecules (1-5 kD) to complexes such as glutamine synthetase and acetylcholine receptor. For particles with spherical or cylindrical symmetry, these programs will also permit determination of mass as a function of radius.

扫描透射电子显微镜（Stem）可以提供低剂量， 未染色的生物样品的高分辨率图像。 孤立的对象 或可以集成离散的图像特征（减去之后 底物）以确定分子量，因此提供直接链接 在生物化学和电子显微镜之间。 茎的准确性 在10 kd时，蜂窝状的重量从约20％提高到100 kd时约4％ 对于成像的单个粒子，高于100万d的1％ 剂量为10 El/Angstroms平方。 tRNA，核小体的实际结果， 纤维蛋白原，RNA聚合酶，谷氨酰胺合成酶，30S和50S核糖体 亚基，丝状病毒和TMV与 理论预测。 平均值的标准错误可以通过 平均超过100-1,000个颗粒。 粒子质量值的直方图可以 用于表征制剂的同质性。 这项技术 已定期应用于各种用户/合作者 过去几年的布鲁克黑文茎生物技术资源的样品。 我们建议继续这些富有成效的合作和服务 活动的同时完善技术的实际方面。 更远 降低样本温度至20 K有望降低质量损失 现在在120 K处发现的0.25％/El Angstroms平方（比300少8倍 k），允许更高剂量和更高的准确性。 表面还原 预计在较低温度下的迁移会导致重新分布较少 辐射损伤期间的质量。 该预测将使用 已知结构的控制样本（例如TMV）。 一个主要的新推力将涉及使用图像平均程序来改进 低剂量图像中的信号与噪声比。 这应该允许扩展 在纤维蛋白原和动力蛋白上已经证明了域质量映射 允许检测小分子（1-5 kd）与复合物的结合 例如谷氨酰胺合成酶和乙酰胆碱受体。 用于颗粒 使用球形或圆柱形对称性，这些程序也将允许 确定质量作为半径的函数。