Correlative Single Particle Imaging Program
Director: Peijun Zhang, with the PCHPI Imaging Core Directors, Zandrea Ambrose and Simon Watkins.
Cryo-electron tomography of intact cells is an emerging technology that is likely to provide a global context for unifying structural data from crystallography / NMR and single molecule approaches, making it possible to determine the spatial arrangements of key proteins and complexes in the cell, with the exciting prospect of being able to follow spatial and temporal changes in these distributions during signaling events. The cryo ET Technology Program is developing and implementing tools for cryo-electron tomography to overcome the current technological barriers and will establish this technique as a standard technology for examining HIV-host cell interactions.
HIV entry can be regarded as an excellent example of a rare, timed event which necessitates the use of fluorescence light microscopy to identify the region of interest (point of entry) for high resolution cryo-electron tomography. The PCHPI has designed and built a microscope stage that allows fluorescence imaging under cryogenic temperature for correlation with tomography. Bright fluorescent tags will be devised for following the virus particles/complexes through the cytoplasm to the host cell nucleus. In addition, novel electron-dense fluorescent labels that are detectable by both fluorescence microscopy and by electron microscopy will be developed. One powerful feature of such labels is their potential for allowing correlative imaging. With these labels, imaging of dynamic, living cells with fluorescence light microscopy can potentially be interfaced with high resolution snapshots provided by cryo-electron tomography.
The PCHPI makes use of the cryo-EM facility in the Department of Structural Biology in the basement of the BST3. The facility houses state-of-the-art electron microscopy instruments and has full capability to carry out high resolution three-dimensional structural analysis of proteins, protein complexes, phages and viruses, macromolecular assemblies, cellular organelles, and bacterial cells. Three electron microscopes are available: a Polara G2 electron microscope operating at 300kv and a Tecnai F20 electron microscope operating at 200kv, both equipped with high-resolution 4kx4k CCD cameras and capable of low-dose cryo-EM and electron tomographic studies. A Tecnai T12spirit, operating at 120kv, handles room temperature specimens and has features for automatic tuning and alignment. The facility is also equipped with a FEI vitrobot for frozen-hydrated specimen preparation and other ancillaries for cryoEM.
See Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography. Jun S, Ke D, Debiec K, Zhao G, Meng X, Ambrose Z, Gibson GA, Watkins SC, and Zhang P. (2011) Structure 19 (11): 1573-1581. PubMed