Single Cell Molecular Biology Through Electron Microscopy

该奖项提供资金，用于测试使用扫描电子显微镜检测所有生物体中发现的微量大分子（DNA， RNA，蛋白质）。提议的测试将使用基本的印迹技术，包括将大分子吸附到硝化纤维或尼龙膜上，然后将其暴露于抗体或其他高度特异性的检测器大分子中，这些大分子本身被标记为金颗粒。即使是一粒金也能在显微镜下清晰可见。原则上，所提出的技术的成功发展将允许在单个细胞中检测少量，可能少到一个或两个目标大分子分子。在过去的二十年里，分子生物学取得了惊人的进步，对特定蛋白质、rna和dna使用一般的、但高度特异性的、容易定量的测定方法至关重要。正如现在所应用的那样，大多数这些测定都适用于从培养物中生长或存在于组织、器官甚至整个生物体中的大量细胞中提取的材料。到目前为止，只有少数高度专业化的测试能够检测到单个细胞中存在的微量此类分子。扩展更一般的技术，以允许分析单细胞中低水平的独特分子，对于理解分化良好的多细胞生物的发育控制的进展至关重要。虽然并非没有风险，但将要采取的办法有可能提供这种延长。

This award provides funds to test the use of scanning electron microscopy for detection of minute quantities of the types of macromolecules (DNA, RNA, protein) found in all living organisms. The proposed test will use basic blotting techniques that involve adsorption of the macromolecule to a nitrocellulose or nylon membrane which is then exposed to antibodies or other, highly specific detector macromolecules that are themselves labelled with gold particles. Even a single particle of gold is readily visualized in the microscope. In principle, the successful development of the proposed technique would permit detection of a small number, possibly as few as one or two, molecules of the target macromolecule in a single cell. The use of general, but highly specific, readily quantitated assays for particular proteins, RNAs and DNAs has been crucial to the astounding progress of molecular biology in the last two decades. As now applied, most of these assays are applied to material extracted from large numbers of cells either grown in culture or present in a tissue, organ or even an entire organism. As yet, only a few, highly specialized tests are able to detect the minute quantities of such molecules present in single cells. The extension of more general techniques to permit analysis of low levels of unique molecules in single cells appears crucial to progress in understanding the control of development of well- differentiated multicellular organisms. Although not without risk, the approach to be undertaken has the potential of providing such an extension.