3D Electron Microscopy Of Molecular Assemblies
Zhang Lab University of Pittsburgh School of Medicine Department of Structural Biology
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Technology development: Correlative microscopy for single particle imaging

We are constantly developing new tools and techniques to overcome the many challenges and to enhance the capability of cryo-EM. One of the current NIH funded projects focuses on cryo-FIB processing of vitreous biological specimen to reduce the sample thickness, thus permitting cryo-EM study of many key biological systems, such as mammalian cells, plant cells, yeasts and tissues. In addition, we are also developing methods for nanoscale single molecule localization in 3D cellular tomograms, including a novel GFP-equivalent genetically taggable EM probe for 3D localization and super resolution microscopy for nanoscale localization of fluorescent labeled proteins in the ultrastructure. Biosynthetic attachable tags that are detectable by both fluorescence microscopy as well as by cryo-EM are most desirable. One powerful outcome made possible by such labels is the potential for correlative imaging of dynamic living cells with fluorescence light microscopy, interfaced with high resolution snapshots provided by cryo-electron tomography.

Jun S., Zhao G., Ning J., Gibson G., Watkins S. and Zhang P.* (2013) Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions. J Vis Exp. 76: e50386, doi:10.3791/50386. 

Strunk K., Wang K., Ke D., Gray J. L. and Zhang P.* (2012) Thinning of large mammalian cells for cryo-TEM characterization by cryo-FIB milling. J. Microscopy 247(3):220-227.

Wang K., Strunk K., Zhao G., Gray J. L. and Zhang P.* (2012) 3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography. J Struct Biol 180(2): 318-326.

Jun S., Ke D., Debiec K., Zhao G., Meng X., Ambrose Z., Gibson G. A., Watkins S. C. and Zhang P.* (2011) Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography. Structure 19(11):1573-1581.

Song C., Zhao G., Zhang P. and Rosi N.L. (2010) Expeditious Synthesis and Assembly of Spherical Gold Nanoparticle Superstructures. J. Am. Chem. Soc. 132(40):14033-14035.

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