The overall goal of the proposed work is to develop general, biologically useful strategies for detecting single molecules or molecular assemblies in living cells with the light microscope (LM) and to enable direct visualization in the electron microscope (EM) of these same molecules that have been tagged, observed and dynamically tracked in the LM. The research activities planned grow from successful development of new fluorescent proteins, quantum dot construction and targeting, tetracysteine labeling techniques for light and electron microscopy, as well as from extensive application of such new techniques to open questions in cell and molecular biology. This proposal includes possible ways to target quantum dots to genetically specified macromolecules and creation of fluorescent proteins with long excited state lifetimes and increased photostability. We also propose to increase the sensitivity of diaminobenzidine (DAB) photoconversion mediated by biarsenical-tetracysteine-tagging, to develop fluorescent proteins that can be photoconverted, and to develop ways to photoconvert two different proteins into the EM equivalent of two colors. Model membrane proteins and intracellular constituents will be studied with sensitive detection devices and optics to evaluate the utility of probe systems being developed. These activities benefit from the combination of this applicant teams expertise in chemistry, imaging instrument development, cell and molecular biology, physics and biophysics.
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