USING NANOGOLD AS A SPECIFIC MARKER IN TOMOGRAPHY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have been investigating the utility of electron dense markers, particularly gold particles, which can be internalized into live cells under physiological conditions. It is important that these markers are as small as possible, so as not to perturb the system or organelle under study. Nanogold clusters can be covalently coupled to any suitable reactive group such as oligonucleotides, lipids, peptides, proteins and others. They are extremely uniform in size (1.4nm) and provide close to stoichiometric labeling. Once internalized, these tiny particles are usually then enhanced with silver to produce highly visible grains 2-20 nm in size. However, we would like to visualize nanogold particles in tomographic reconstructions without the addition of silver enhancement. For these experiments we either applied nanogold to the surface of epoxy sections containing biological material which had not been previously post stained with heavy metals, or attempted to internalize the nanogold into samples prior to embedding them in resin. Sections (150nm) were imaged at 59,000X in the Technai F30 microscope and tilt series were taken at 1 degree increments over 120 degrees with a pixel size corresponding to 0.39 nm. In the resulting tomographic reconstructions, we were able to clearly see nanogold lying at the section surface. However, nanogold internalized into live cells was not visible, likely because we had not located areas of internalization. We have attempted to use the dark field mode as well as the Gatan Energy Filter to locate nanogold within sections of biological material prior to collecting tilt series data, but these trials were not successful. Our efforts suggest that we must modify our methods of imaging to locate nanogold, either an electron optical method, like STEM dark-field imaging, or a chemical method, like silver enhancement carried out during freeze-substitution. We have, therefore, worked on methods to enlarge nanogold with silver in the presence of organic solvents at low temperature, during freeze substitution. Experiments using nanogold suspensions applied to EM grids, followed by combinations of silver enhancement chemicals dissolved in acetone have been successful in producing particles of 3-5 nm. We have now begun to apply this technology to an in vivo system. The Bjorkman lab has conjugated nanogold particles to IgG and have applied the conjugates at low pH to cultured monolayers of MDCK cells that have been grown under conditions in which the cells form sheets that resemble polarized epithelia. The same reagent has been administered to the intestine of newborn rats Following preparation by rapid freezing we have begun to add silver enhancement reagents to the freeze substitution protocol at -50oC. Enhanced nanogold particles may now be tracked through the vesicular compartments on the apical side of the cells.