Cryogenic-temperature transmission electron microscopy (cryo-TEM), namely TEM imaging of liquids ultrafast cooled to cryogenic temperatures has become an indispensable tool to acquire high-resolution direct-images of self-aggregating liquids, containing features on the nanoscopic scale. A wide range of systems of low- and high-molecular weight solutes, synthetic and biological, has been studied by the technique. While most cryo-TEM work has been done on aqueous systems, more recently the technique has been extended to non-aqueous solvents as well.
The term ‘cryo-TEM’ actually refers to two techniques: direct-imaging cryo-TEM, by which a thin vitrified sample is examined by the TEM at cryogenic temperatures, and freeze-fracture-replication cryo-TEM (FFR), by which a carbon-metal replica of the fractured fast-cooled specimen is examined at room temperature by the TEM.
Direct-imaging cryo-TEM allows in situ imaging of delicate structures of soft matter, including liquid systems, providing unique information not obtainable by other methods. Cryo-TEM can provide high-resolution images of complex fluids in a near in situ state. Samples embedded in a thin layer of vitrified solvent do not exhibit artifacts that would normally occur when using chemical fixation or staining-and-drying techniques. Cryo- TEM has been useful in imaging biological molecules in aqueous solutions.
Cryo-TEM technique involves production of frozen hydrated specimens prepared, maintained and observed within a layer of vitreous ice. Below 273 K, ice may exist as hexagonal or cubic crystals, or as the vitreous form, which is essentially a supercooled liquid. Vitrification occurs when cooling is very rapid (~105 degrees). Vitrification of a thin specimen is usually achieved by rapid plunging it into liquid hydrocarbon, mostly ethane (an efficient cryogen), held at the liquid nitrogen temperature. This helps to avoid formation of ice crystals and thus the damage they cause, allowing the investigation of a specimen in the fully hydrated, close to physiological form. The vitreous state persists if the temperatures are maintained below the devitrification level (~130 K). Specimens can effectively be observed in the absence of the background noise contributed by normal support films.
Freeze-fracture-replication (FFR) is an indirect way to get an image from a specimen, meaning the image is obtained from a replication of the sample and not from the sample itself. This method includes few steps of preparation (see figure):
1. Freezing – the sample is cooled (in rapid cooling) to a very low temperature
2. Fracturing – the top part of the sample is cracked open
3. Etching – by sublimation of the ice that on the sample (not necessary step)
4. Replication – the surface of the sample is coevered with heavy metal (e.g. Platinum) by deposition at an angle of 45ยบ or less to the horizon, to enhance contrast (“shadowing”). Above it, a carbon layer is added for mechanical stabilization of the replica.
5. Dissolving the specimen – the replica is floated onto the surface of a powerful solvent to dissolve away the specimen.
6. Washing – the replica is washed and picked up on a cooper grid for examination in the cryo TEM.
Scheme of the different stages preparation of the replica
This technique provide image of the topography of the specimen, fine imaged and fine particles can be imaged. System with high viscosity, or that containing large particles that cannot be accommodated in the thin specimen of direct imaging cryo-TEM, cannot be examined in direct-imaging cryo-TEM, but can be examined in the FFR technique.
