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. Tomographic reconstruction of frozen-hydrated biological samples, embedded in amorphous ice, is a powerful method for the study of cell structure in its initially native condition. However, few biological samples are less than 0.5 micrometers along the beam axis, thin enough to be viewed in their entirety with 300 KeV electrons. It is therefore important to develop methods for slicing cells while they are frozen. A diamond knife in a good cryo-ultramicrotome can cut the desired sections without warming them to temperatures at which amorphous ice will crystallize, but stable diamond knives are thick (the angle included at the knife edge is 25-35 degrees), so the amorphous ice is usually compressed and sharply bent by the cutting process. To solve these problems we are investigating the use of carbon nanotubes (CNTs) as devices that could be used as tight wires to sever thin slices from a block of vitrified biological material. Single walled CNTs have a radius of about 1 nm, less then the curvature at the edge of a sharp diamond knife. CNTs can be millimeters long and are known to be strong, but how strong relative to amorphous ice remains to be determined. We have examined commercially obtained and homegrown CNTs in a scanning electron microscope, disentangled from their neighbors by micromanipulation. We stretch them over a thin loop of tungsten wire and have developed a 'welding' process that forms a strong bond between the CNT and the tungsten. We are working to streamline this process so it can be used to make useful numbers of CNT-based cutting devices. We are also experimenting with devices that can measure the hardness and viscosity of vitreous ice, so we can understand the physical requirements for the microtomy we plan.
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| 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 |
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