We seek support for the development of technologies that will help elucidate the three-dimensional (3-D) fine structure of cells and tissues. Two approaches to imaging will be employed: (1) thick samples of biological material will be studied in a JEM-1000 high voltage electron microscope (HVEM), and serial tilted views will be imaged to provide information about the 3-D structure of the specimen; (2) serial sections of various thickness will be imaged either in the HVEM or by conventional electron microscopy and assembled into 3-D models by a combination of image processing and computer graphics. The major thrust of our efforts will be to develop specimen preparation technologies for these imaging approaches. (1) We will continue our work on structural neurobiology, improving existing methods for the selective staining of specific cells and molecules. Tomographic methods will be combined with stereo modeling and serial section reconstruction to extract quantitative information about neuronal geometry. (2) We will investigate the utility of high pressure rapid freezing for the study of specimens too large to be well preserved by conventional freezing methods. Cryofixed material will then be treated by several secondary methods designed to permit reliable visualization of fine structure and of the location of specific antigens. (3) We will explore the utility of cryoHVEM for the study of frozen hydrated material, using flagella and basal bodies as test systems. (4) We will continue our development of methods for in situ hybridization at the EM level to reveal the location of nucleic acids with biotinylated probes.
| 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 |
| Höög, Johanna L; Lötvall, Jan (2015) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J Extracell Vesicles 4:28680 |
| 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 |
| 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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