A new class of immunolabels will be developed that comprise a number of fluorophores attached to a gold cluster complex. These will then be covalently, site-specifically coupled to antibodies and proteins to give a range of bifunctional immunoprobes with improved sensitivities, retention of immunoreactivity, and penetration up to 30 microns into cells and tissue sections. Correlation between two complementary sets of data will result from conducting two experiments with a single probe: 1) three dimensional fluorescence imaging of nuclear structures and dynamic cell processes; and 2) electron microscopy of molecular organization at the ultrastructural level. Multiple fluorescent groups will be coupled to a single gold cluster while their hydrophobicity is compensated by other hydrophilic groups, giving probes with sensitivities five times or more greater than those currently available. They will be evaluated by immunoblotting, and use to study three systems by fluorescence and electron microscopy; 1) coordination between components of nuclear mRNA splicing sites; 2) organization of individual proteins of the sodium/potassium and sodium/calcium pumps in smooth muscle cells; and 3) interphase chromosome structure and nuclear architecture, using microinjected fluorescent/gold labeled DNA-binding proteins and stains to study dynamic processes in living cells.
| 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 |
| Powell, R D; Halsey, C M; Spector, D L et al. (1997) A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy. J Histochem Cytochem 45:947-56 |
