A new type of immunoprobe will be developed, containing both an enzyme and a covalently linked 1.4 nm, 3 nm, 5 nm or 10 nm gold label. These will be used for correlative microscopic staining of biologically significant targets at the cell and tissue level by fluorescence or brightfield light microscopy using either a fluorescent or chromogenic substrate, and at the macromolecular level by electron microscopy, using a single labeling procedure. By enzymatic deposition of a fluorescent substrate, the new probes will enable combined immunofluorescent labeling and 3, 5 or 10 nm gold labeling, a procedure previously prohibited by the strong fluorescence quenching properties of the gold particles. Correlation of the two complementary data sets will be improved by the elimination of the effects on specimen morphology of processing between labeling experiments. In addition, these probes will facilitate multiple labeling in which two or more targets are distinguished by different colors at the light or fluorescence level, and different sized gold particles at the electron microscopic level. A variety of synthetic cross-linking rationales will be used to prepare probes, and labeling performance of each component of the resulting probe will be carefully compared with the equivalent singly labeled conjugate using blots, light microscopy on cell and tissue control slides, and electron microscopy. Probes meeting targets for sensitivity, specificity and specimen penetration will be evaluated in two research applications: (i) localization of polar tube proteins in microsporida, and (ii) structural and localization studies on pre-mRNA transciption and splicing factors in the mammalian cell nucleus. This research will provide researchers with new tools for combining data on normal and disease processes in biological systems at the cellular and molecular level, enabling the acquisition of fundamental new knowledge of how these processes work that will support the development of improved dignostics and therapeutics. In addition, it is anticipated that the new probes will be capable of higher detection sensitivities than either detection component used alone, and this may be used for the earlier detection of cancer or other genetic abberations through improved molecular diagnostic procedures such as DNA and RNA in situ hybridization and immunohistochemistry of low abundance targets.
