We have obtained a side entry cold stage for the HVEM and are using it to explore the efficacy of high voltage electrons for imaging cells in a frozen hydrated condition. PtK cells have been used as a test specimen to work out methods for plunge freezing cells with no detectable damage from freezing or from drying before freezing. Well frozen, frozen-hydrated cells display sufficient contrast at appropriate levels of defocus to reveal the familiar organelles with surprising clarity. In the region of the cell nucleus, the cell is too thick for informative imaging, but near the periphery, both the large organelles like mitochondria and some elements of the cytoskeleton are clearly visible. Indeed, at the cell's margin, single microfilaments can be distinguished. This technology is now being applied to cultured 3T3 cells to characterize the ultrastructure of the leading lamina. We are exploring limited tilt strategies and dose fractionation to obtain tomograms of the leading edge to understand the 3D arrangement of microfilaments in this region. By using the material that has been prepared simply by fast freezing, we hope to learn the details of the structural changes that occur during the cell migrations.
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