Structure of the cytoplasmic filament system in freeze-dried whole mounts viewed by HVEM

Freeze-drying (FD) was used as an alternative to critical point-drying (CPD) for the preparation of whole mounts to study the cytoplasmic filament system in mammalian cultured cells by high voltage electron microscopy (HVEM). Rapid quenching methods such as plunging a grid into liquid propane cooled by LN2 or collision with a clean surface of a copper block cooled to LHe temperature were used to avoid ice crystal formation. For freeze-drying, a special apparatus was built that allowed the specimen to be kept at 145 K for 2-3 days at a vacuum of about 2 X 10(-7) Torr followed by gradual stepwise warming to room temperature. Purified skeletal muscle actin served as a test object for which the structure was known through independent techniques. PtK-1 cells, grown on gold grids and fixed in buffered glutaraldehyde, were used to study the structure of the cytoplasmic filament system. Under conditions that should avoid formation of ice crystals, the structure of actin fibres was as expected on the basis of previous studies, i.e. a tangle of independent uniform filaments about 7 nm thick, Using similar conditions, the cytoplasmic filaments of PtK-1 cells were equally distinct and evenly thick along their length and were usually associated with particles about 10 nm thick. On the other hand, when the sublimation time at the low temperature was cut short, both purified actin and the cytoplasmic filament system formed a network of tapering filaments devoid of particles, similar to that which has been described as a 'microtrabecular lattice'. We therefore conclude that when this structure is seen in FD preparations, it is probably the result of distortion produced by faulty FD procedure.