Hypobaric helium atmospheres for light and electron microscopy of mammalian cells

Abstract

SUMMARY 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°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°C. No 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 The filopodia, or microvilli attaching the cells to the substrate, became withdrawn, with only occasional retraction fibres remaining. Blebs appeared around the cell surface and increased in size as hypobaric exposures progressed. Vacuoles, or clear 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× increase in size as they respread on the substrate. Concurrently, the cytoplasm appeared oedematous and the cytoplasmic granules exhibited Brownian‐like motion. If hypobaric exposure was continued much beyond this latter stage, the cells were irrecoverable and the majority died after lysis of the cell membrane. It is apparent from this and other studies that mammalian cells begin to exhibit signs of stress when held at pressures below 300 Torr. This stress can lead to rapid killing of the cells by membrane lysis at lower pressure. A moist helium atmosphere of 300 Torr should permit at least 45% beam transmission in a conventional transmission electron microscope at 200 kV and should prove to be a major constituent of a supportive atmosphere suitable for maintaining cell viability and normal morphology during electron microscopy of living cells. The other major parameter (apart from radiation damage) for good quality electron imaging of living cells exhibiting various forms of motility and intracellular movement, is that of the depth of the surrounding aqueous medium. The possibility exists that media layers sufficiently deep to prevent immobility caused by surface tension effects, may prove to be a limiting factor for successful electron imaging.