Electron microscopy of frozen water and aqueous solutions

Abstract

SUMMARY Thin layers of pure water or aqueous solutions are frozen in the vitreous state or with the water phase in the form of hexagonal or cubic crystals, either by using a spray‐freezing method or by spreading the liquid on alkylamine treated films. The specimens are observed in a conventional and in a scanning transmission electron microscope at temperatures down to 25 K. In general, the formation of crystals and segregation of solutes during freezing, devitrification and evaporation upon warming, take place as foreseen by previous X‐ray, thermal, optical and electron microscopical studies. Electron beam damage appears in three forms. The devitrification of vitreous ice. The slow loss of material for the specimen at a rate of about one molecule of pure water for every sixty electrons. The bubbling in solutions of organic material for doses in the range of thousands of e nm−2. We propose a possible model for the mechanism of beam damage in aqueous solutions. The structural and thermal properties of pure frozen water important for electron microscopy are summarized in an appendix.

Single particle electron cryomicroscopy: trends, issues and future perspective

“…They characterised vitreous, cubic and hexagonal forms of ice, and developed a simple plunge-freezing method for preparing grids Dubochet et al 1982Dubochet et al , 1984. Their work depended on the development of the first ultra-high vacuum specimen environment in the Philips 400 electron microscope, which had been required by the materials science community to avoid contamination of their clean metallic specimens.…”

“…Following the preparation of samples for cryoEM using the method of Dubochet et al (1982Dubochet et al ( ,1984, we typically apply a small drop (3 µl) of a solution of the biological structure of interest to a holey carbon film on a metal EM grid. This is then blotted for a few seconds (using filter paper) and rapidly frozen by plunging the grid into liquid ethane cooled by liquid nitrogen to a temperature just above the freezing point of liquid ethane (−183°C).…”

Magnification calibration and the determination of spherical virus diameters using cryo-microscopy

“…Diffraction pattern films were digitized as described in section 2.2 at two raster settings (25 µm and 100 µm) to allow accurate measurement of both the strong 1.15 nm 6th layer line of TMV and the [111] 0.366 nm diffraction ring of cubic ice [43].…”

“…The spacing of layer lines in the helical diffraction pattern of TMV was calibrated with the powder diffraction rings of the 0.366 nm [111] Bragg reflection of cubic ice [43]. The average pitch of the 1-start helix of TMV (obtained from three different diffraction patterns) was determined to be 2.28 nm.…”

“…The remarkable agreement between dimensions of aqueous samples measured by X-ray diffraction and frozen-hydrated specimens measured by electron microscopy is somewhat surprising since water expands 2-3% upon freezing to the vitreous state. One might expect such expansion to create forces which would act to compress the biological sample [43]. Apparent resistance of the sample to structural change caused by vitrification could indicate that water redistributes during freezing or that differences exist between the density of vitreous water in the free solvent compared with the hydration shell surrounding the biological molecule [29].…”