We have revived our Facility's interest in imaging whole mounts of frozen cells by following two approaches: seeking better methods for culturing and freezing cultured mammalian cells; and looking for ways to fix these cells by freeze-substitution so they may be embedded in plastic resins for tomographic analysis in the HVEM. The ultimate goal for the first approach is tomographic reconstruction of frozen hydrated cells, but the large number of images required for such work, together with the radiation sensitivity of frozen-hydrated material, makes this approach difficult. We hope that our new CCD camera will help considerably in such work, once it has been optimized, but experience so far suggests that the resolution of such 3-D reconstructions will be limited by radiation damage. Plastic embedding produces specimens with a significant increase in resistance to radiation damage. Our second approach is therefore to get well frozen cells, freeze-substituted and embedded into hard plastics that are no thicker than the cell itself. Subsequently, whole mount imaging of these preparations should provide data for tomographic modeling of specimens that are more robust. Preliminary data show that cells can be frozen well by culturing them on carbon-stabilized, formvar-coated gold grids and plunging them into liquid ethane. Some of these have been fixed by freeze-substitution in OsO4 in acetone at -90oC, then embedded in different resins. These resins were applied directly to the grid face and blotted to an appropriate thinness. LR White cured at room temperature has provided the best thin embedment, and we will proceed with tomographic study of these cells. S T10
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