BHK cells infected with the Semlike forest virus are being studied using semi-thick sections and the IVEM. There are two goals of this TRD project. The first is to reconstruct virus core particles in the cytoplasm of tissue culture cells. The second is to study the trans-membrane budding process of the core particle. We consider this a TRD project because we are using the virus as a test specimen to determine the maximum resolution that can be obtained by tomography on plastic sections. The core particles are 40nm and the complete particles are 70nm. Thus, to be sure most particles are not truncated, sections on the order of three times the particle diameter are needed. In previous work, we have been unable to clearly identify the core particles in the cytoplasm because they were very few, although budding particles in and on the cell membrane could be seen. Over the past year a new mutant has been developed, in which the virus particles are very numerous in the cells, although no budding is seen. Thus we now have the two types of specimen we need. In previous work, we could not obtain sufficient resolution of the core particles to make reconstructions, presumably due to conventional specimen preparation and staining. The new material has been prepared by high-pressure freezing and freeze-substitution, without the use of osmium. Post-stained sections of the new material looks promising. Study of the budding process by electron tomography is straightforward. We are now concentrating on determining if we have enough resolution to do rotationally-averaged reconstructions of the core particles. Post-staining of the sections may decrease the resolution, or plastic embeddment itself might cause a problem by contrast-matching or granularity. To investigate this issues, we have arranged to record some test images on a 200kV field-emission TEM with energy-filtered imaging, which is in the lab of Dr. T. S. Kuan of SUNY Albany Physics Dept.
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