Hypobaric helium atmospheres for light and electron microscopy of mammalian cells

Abstract

The development of electron microscope environmental/hydration chambers (EMC's) for the examination of living biological specimens has necessitated a search for a suitable gaseous environment that is compatible with both electron imaging and cell survival. Helium has a lower electron scattering cross section than air. The effects of hydrated helium and hydrated air atmospheres in an EMC at pressures 50-200 Torr were examined with a JEM 200 transmission electron microscope operated at 200 kV and 22 degrees C. Results showed that whereas 200 Torr of air reduced beam transmission by more than 90% under these conditions, 200 Torr of moist helium only reduced beam transmission by 30%. The effects of sub-atmospheric or hypobaric helium atmospheres on human white blood cells attached to glass coverslips were investigated by phase-contrast light microscopy in a simulated EMC or light microscope environmental chamber (LMC) at ...37 degrees C. gross morphological changes were observed in cells held at pressures of 300 Torr of moist helium for exposure times of 15 min. At pressures below 300 Torr, morphological changes occurred more rapidly as the pressure was reduced. At the lowest pressures tested (60 and 80 Torr) 50% of the cell populations died within 6-7 min. The development of morphological changes produced by hypobaric stress followed a characteristic pattern. The filopodia, or microvilli attaching the cells to the substrate, became withdrawn, with only occasional retraction fibers remaining. Blends appeared around the cell surface and increased in size as hypobaric exposures progressed. Vacuoles, or cler spherical regions, appeared within the cytoplasm and the overall shape of the cells became circular. The cells became flatter during circularization, showing an apparent 2-2-5 X increase in size as they respread on the substrate...