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 are working to improve the quality and reproducibility of hydrated cryosections, cut from blocks of high-pressure frozen (HPF) tissues or cells, so as to prepare specimens for cryoelectron microscopy. Blocks of samples are prepared so they remain domed in the flat half of a two-piece brass HPF hat. The hat is then mounted in a brass collet that fits into the specimen chuck of a Leica UltraCut-UCT/FCS cryomicrotome. The block is trimmed and faced with a custom Diatome diamond trimming tool, and sections are cut with a Diatome Cryo-P diamond knife. Our best knife has a 25o included angle at the knife-edge. The diamond knife itself is a significant contributor to two types of artifact common to hydrated cryosections; compression of the section parallel to the cutting direction and fracturing of the section as it bends away from the knife edge. These fractures appear as ripples in the section surface when it is viewed in the electron microscope. A major goal of this project is to reduce the surface fracturing of sections. Although we have not eliminated this artifact, we have reduced it significantly by addressing several factors other than the knife angle. These include reducing the size of the specimen block face, shaping the face as a vertical rectangle rather than as a horizontal trapezoid, adjustment of section thickness and close control of static within the sectioning chamber. Furthermore, we have found that pellets of yeast yield sections of much higher quality and reproducibility than pellets of mammalian cells; when mammalian cells are mixed with an equal volume of yeast prior to HPF, the resulting cryosections are similar in quality to the pure yeast samples. Close inspection of vitreous sections of yeast cells revealed that the cell wall (especially the inner most layer) showed a significant reduction in the surface fractures, or no fracturing at all. We are therefore exploring the use of molecules that serve as major cell wall components, such as beta-glucans, as freezing media in place of standard cryoprotectants. We will also investigate the extent to which the temperature of the sectioning chamber can be varied in order to provide an optimal balance between section quality/reproducibility and sample preservation.
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| 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: |
| 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 |
| 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 |
| 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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