The purpose of this study is the development of a new labeling technology for use with electron microscopy that will permit investigators to rapidly, reliably, and simultaneously identify and localize multiple labeled molecular species in a single specimen. This is in response to PA-00-084 """"""""Tachnology Development for High Resolution Electron Microscopy."""""""" Priorities for this PA include """"""""analysis of spatial distribution of macromolecules in cells,"""""""" and """"""""..better reagents for.. The specific labeling and detection of macromolecules in cells."""""""" A common requirement of high resolution studies is the localization and co- localization of multiple molecular species. Light microscopic based imaging systems solve this problem, in part, through the use of flourescent dyes with differing emission wavelengths. The different colors serve to unambiguously and simultaneously identify several different labels. Labeling systems which permit the simultaneous identification of multiple protein species at molecular levels of resolution are required now for electron microscopic studies. Techniques currently available for EM level co-localization, employ electron """"""""dense"""""""" colloidal gold nano-particles of differing sizes. Unfortunately, to ensure that there is no overlap in particle size, generally only one or two particle sizes within the resolution range needed can be employed at once. Steric problems arise because of the substantially greater size of the larger particle and differences in the case of detection in differently sized labels also occurs. Hence the number of different molecular species which can be identified and localized relative to one another is extremely limited. Studies proposed here will employ two new approaches. The first approach is to synthesize colloidal nano-particles of the same size but of multiple different shapes which are readily distinguishable in scanning electron microscopy and in transmission electron microscopy. The second approach is the synthesis of and use of colloidal nano-particles of similar size but of different elemental composition. The Energy Filtering Transmission Electron Microscope will be employed to differentiate the particles of different composition based on the electron energy loss spectra associated with the particular elemental composition. The two approaches, singly or in combination, encompass a direct and rapid methodology which permit simultaneous identification of a substantial number of different molecular species or specific epitopes within individual molecules at high levels of spatial resolution.
| Meyer, Daryl A; Oliver, Julie A; Albrecht, Ralph M (2010) Colloidal palladium particles of different shapes for electron microscopy labeling. Microsc Microanal 16:33-42 |
| Kandela, Irawati K; Bleher, Reiner; Albrecht, Ralph M (2007) Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles. J Histochem Cytochem 55:983-90 |
