Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage...

We carried out a unique comparative study between three modes of cryo-scanning electron imaging: high-vacuum, low-voltage and low-vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< -110 degrees C) or undergoes sublimation (-110 to -90 degrees C). At high magnification, high-vacuum imaging of coated specimens gave the best results for 'static' specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight 'classical' charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low-vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti-contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.