This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
| Giddings Jr, Thomas H; Morphew, Mary K; McIntosh, J Richard (2017) Preparing Fission Yeast for Electron Microscopy. Cold Spring Harb Protoc 2017: |
| Zhao, Xiaowei; Schwartz, Cindi L; Pierson, Jason et al. (2017) Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium ""Candidatus Pelagibacter ubique"". Appl Environ Microbiol 83: |
| Brown, Joanna R; Schwartz, Cindi L; Heumann, John M et al. (2016) A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc. J Struct Biol 194:38-48 |
| Saheki, Yasunori; Bian, Xin; Schauder, Curtis M et al. (2016) Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nat Cell Biol 18:504-15 |
| Höög, Johanna L; Lacomble, Sylvain; Bouchet-Marquis, Cedric et al. (2016) 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction. PLoS Negl Trop Dis 10:e0004312 |
| Park, J Genevieve; Palmer, Amy E (2015) Properties and use of genetically encoded FRET sensors for cytosolic and organellar Ca2+ measurements. Cold Spring Harb Protoc 2015:pdb.top066043 |
| McCoy, Kelsey M; Tubman, Emily S; Claas, Allison et al. (2015) Physical limits on kinesin-5-mediated chromosome congression in the smallest mitotic spindles. Mol Biol Cell 26:3999-4014 |
| Höög, Johanna L; Lötvall, Jan (2015) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J Extracell Vesicles 4:28680 |
| Marc, Robert E; Anderson, James R; Jones, Bryan W et al. (2014) The AII amacrine cell connectome: a dense network hub. Front Neural Circuits 8:104 |
| Weber, Britta; Tranfield, Erin M; Höög, Johanna L et al. (2014) Automated stitching of microtubule centerlines across serial electron tomograms. PLoS One 9:e113222 |
Showing the most recent 10 out of 84 publications
