Probes will be prepared, based on the 1.4 nm Nanogold cluster label, for the correlative fluorescence and electron microscopy of living cells, which may be transfected without requiring physical or chemical disruption of the cell membrane for transfection. Three approaches will be investigated: preparation of membrane permeable analogs of combined fluorescent and Nanogold antibody probes either by the incorporation of permeabilizing chemical groups such as acetoxymethyl, or by the conjugation of peptides corresponding to protein transduction domains such as the HIV-1 Tat peptide to facilitate delivery through cell membranes; preparation of membrane permeable gold cluster probes targeted to green and other colored fluorescent proteins by means of antibodies or nitrilotriacetic acid groups which target fused polyhistidine tags; and conjugates of Nanogold with FlAsH or with its corresponding binding peptide targeted respectively to the expressed peptide or to FlAsH-labeled targets. The new probes will be evaluated in the correlative localization of genes, RNA and expressed proteins in the mammalian cell nucleus, and to study the large-scale chromatin structure in the nucleus.
