Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution

Investigations of cellular processes demand immediate arresting of the proc ess at any given time and excellent retention of cellular material and exce llent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-co ated sapphire disks. For cryofixation the sapphire disks covered with a cel l monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the c ryogen was prevented by using a partially insulated cylinder and by vigorou s stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guill otine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at h igh contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temp erature was raised to diverse final temperatures. Substitution for 4 hours at -90 degreesC in anhydrous acetone containing 0.25% anhydrous glutaraldeh yde and 0.5% osmium tetroxide followed by a temperature rise of 5 degreesC/ hour to 0 degreesC and final incubation for 1 hour at 0 degreesC resulted i n high contrast and excellent visibility of subcellular components at the l evel of the membrane bilayer. The high spatial and temporal resolution make s this methodology an excellent tool for studying cell membrane-bound proce sses, such as virus-cell interactions. (C) Wiley-Liss, Inc.