Covalently linkable 3-15 nm gold reagents will be prepared stabilized with formulations that incorporate a hydrophobic chelating thiol domain for stabilizing the gold nanoparticles, and hydrophilic surface groups that provide a wide choice of surface properties and conjugation reactions. Methods are proposed for electron microscopic localization of targets following in vivo and on section-ex vivo gold-labeling in living cells and in well preserved tissues respectively. Clonable expression tags will be used in which yellow and green fluorescent proteins (YFP and GFP respectively) are expressed in combination with hexahistidine tag (His-tag) and lac repressor respectively. The fluorescent protein, His-tag and lac repressor will then be targeted by biocompatible, non-toxic covalently linkable gold nanoparticles functionalized with antibodies or nitrilotriacetic acid (NTA)-Ni(II), a chelate which binds to His-tags with comparable strength and specificity to antigen-antibody binding. In phase I, the use of the 10 and 15 nm gold-NTA-Ni(II) tag will be optimized by modification of the gold surface for highest possible compatibility and stability for on-section labeling while 3 and 5 nm gold covalently linked to anti-lacI or anti-GFP antibodies will be optimized for in vivo gold labeling using recently developed microinjection technique. Finally, the relation between gold size, thickness of the protective shell and protein fluorescence will be investigated in order to determine whether gold particles give fluorescence quenching or enhancement, and the spacing between the fluorescent species and labeled expression tags adjusted, if necessary. The proposed labels can be directly visualized in the transmission electron microscope without the need for signal enhancement by silver or gold and will enable correlative light and electron microscopy of cellular targets.
This project will provide researchers in cell, molecular and structural biology with methods and simple, universal reagents for electron microscopy labeling conducted in parallel with lac repressor or poly-histidine tags fused with colored fluorescent protein expressed in cells, and other models like zebrafish. This will also enable the study of dynamic cellular life processes and disease processes in living cells at macromolecular resolution, providing structural information at the molecular level for any system in which genetically engineered fused tags such as, fluorescent fusion proteins, His-tags and lac operator repeats, to yield fundamental insights into a wide variety of systems. This will be used for both, electron microscopic localization in living cells, and for high resolution correlative gold labeling.
| Joshi, V N; Mitra, D; England, M D et al. (2010) Large Covalently Linked Fluorescent and Gold Nanoparticle Immunoprobes. Microsc Microanal 16:966-967 |
