This Fast Track application revolves around the development of new probes that combine multiple fluorophores and large metal cluster complexes. These unique probes are to be developed for dual use in electron and fluorescence microscopy. Several new probes are proposed for the development during Phase I: 1) large platinum and palladium cluster complexes (1.8-3.6 nm) that are covalently linked to multiple fluorescent entities and to Fab' antibodies; 2) Fab' antibody conjugated Nanogold clusters (1.4 nm) that will be linked to multiple fluorophores via starburst dendrimer polymers, to eliminate fluorescent quenching problems associated with previous attempts to develop fluorescent gold particles; 3) fluorescent Nanogold and larger clusters that incorporate nickel (II) chelate that binds polyhistidine sequences engineered into His- tagged recombinant protein expressed in cells; 4) Fab' anti-green fluorescence protein (GFP) antibodies conjugated to Nanogold and larger metal clusters for detection of GFP-chimeric proteins expressed in cells; and 5) fluorescent Nanogold and larger clusters that are coupled to dUTP and dATP for enzymatic incorporation into DNA and used for in situ hybridization. In the initial phase, anti-IgG antibody-conjugated probes would be evaluated by immunoblotting serially diluted IgG (with silver enhancement) and as secondary reagents for immunofluorescence labeling of surface red blood cell antigens. The fluorescent and metal cluster-Fab's would also be used as secondary probes to detect snRNPs labeled with monoclonal antibodies and evaluated by confocal and electron microscopy. Fluorescent metal cluster-DNA probes would be tested for ability to detect pre-mRNA transcripts of c-fos and results compared to current applications. Anti- GFP directed probes would be tested against several GFP-chimeric proteins that are expected to have distinct intracellular localization patterns. Phase II would follow with successful application of the new reagents to study three systems by fluorescence and electron microscopy: (i) structure and function of nuclear RNA processing sites; (ii) macromolecular structure of the Na+/K+ and Na+/Ca++ pumps in smooth muscle cells; and (iii) interphase chromosome structure and nuclear architecture, using microinjected dual-probe labeled DNA-binding proteins to study dynamic processes in living cells.
The products have excellent potential for use in the research community, particularly for high resolution electron microscope observations aimed at defining molecular organization. The small size of the probes will limit usefulness unless at high magnification. However, the covalent attachment of probes to primary reagents should reduce nonspecific labeling that is typically a problem in routine labeling with colloidal gold. The potential to develop kits for users to label their own products that contain thiols or amines is an especially attractive feature of the proposal.
| Powell, R D; Halsey, C M; Liu, W et al. (1999) Giant platinum clusters: 2-nm covalent metal cluster labels. J Struct Biol 127:177-84 |
| Powell, R D; Halsey, C M; Hainfeld, J F (1998) Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy. Microsc Res Tech 42:2-12 |
