Sigworth Laboratory
Yale
School of Medicine Cryo-EM Technologies Spherical reconstruction  Spherical reconstruction is a technique we are pursuing for the imaging of membrane proteins reconstituted into lipid vesicles. The idea is to exploit the geometry of small, spherical lipid vesicles both to aid in the determination of orientation angles and to allow compensation for the membrane density in electron micrographs. You can read our first paper on the method: Jiang et al., 2001. -Qiu-Xing Jiang and David Chester |
Tethering surface Obtaining EM images of vesicles has been difficult because the preparation of the cryo-EM specimen involves a blotting step that removes nearly all vesicles from the EM grid. We have been developing an "affinity surface" that provides tethering sites for vesicles. This is an ultra-thin carbon film to which is adsorbed a low density of polyhistidine molecules. In the presence of Ni2+ the polyhistidines form tethering sites for vesicles doped with NTA lipids. -David Chester |
| Holey carbon films Cryo-EM specimens are typically supported by a carbon film containing holes on the order of 1 micron diameter. Best is to have uniform-sized holes in a regular array. We are developing a simple "rubber stamp" technique for patterning the holes. The stamp is a block of PDMS molded from a silicon micromachined master. It is used to transfer a patterned plastic film to a glass surface. The film is transferred t  o metal grids and forms a substrate for the evaporation of carbon.-David Chester and Kate Klemic Algorithms We are working on algorithms for automatic particle selection in micrographs and for real-time image acquisition in the electron microscope. Our approach is a classical multi-channel matched filter. We make use of a reduced-dimension representation of the references for computational efficiency. -Fred Sigworth, Liguo Wang and Shirley Wang. |
Last modified: June 21, 2004 ns.
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