Single Cell Molecular Biology Through Electron Microscopy

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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.