Representative characterisation of inorganic materials using cryo-electron microscopy

Nanoparticles have become increasingly common in a wide variety of application areas from medicine to materials science due to their appealing properties. Nanoparticle characterisation require the use of multiple techniques to analyse physical and chemical properties; among these techniques, electron microscopy is an essential high-resolution imaging technique that is routinely used to inspect nanoparticle size and agglomeration. However, high-resolution analysis of aqueous soft materials such as nanoparticle dispersion, hydrogels, and emulsions is often challenging because conventional dry sample preparation techniques can introduce
artefacts affecting particle agglomeration and the overall structure of the sample. Cryogenic sample preparation and cryogenic electron microscopy are extensively used in the life sciences but are not as developed for their application to inorganic samples.

This thesis aims to representatively characterise inorganic materials using cryo-electron microscopy, employing a range of techniques found in biology and materials science including spectroscopy techniques, tomography, and dark field imaging. Cryo-transmission electron microscopy and cryo-scanning electron microscopy were used for the imaging of inorganic nanoparticles and Pickering emulsions. The spherical 3D structure of Pickering emulsions was seen to be preserved following cryogenic sample preparation using plunge-freezing, which was found suitable for the preparation of sample droplets up to ~600 nm in diameter, which were then analysed using cryogenic transmission electron microscopy, energy dispersive X-ray spectroscopy, and electron energy loss spectroscopy. The 3D nature of frozen hydrated samples allowed for the application of cryo-electron tomography, which was used to successfully visualise nanoparticles and emulsion droplets in 3D space, giving insight on the 3D position and shape of the particles. Lastly, spectroscopy techniques were used together with cryogenic electron tomography to obtain 3D elemental information of the oil and water phases in the emulsion.

Electron beam-induced damage to the sample and ice thickness remain the largest limiting factors to the cryo-transmission electron microscopy imaging of Pickering emulsions, affecting especially the size of emulsion droplets that can be analysed through tilt tomography without encountering issues with low signal-to-noise ratio (at low electron fluence) or sample damage (at high electron fluence). The imaging of sections from Pickering emulsion samples with large droplets (>1 μm) was possible using cryo-focused ion beam-scanning electron microscopy, with the focused ion beam being used for both the 3D imaging of large volumes and the milling a frozen lamella that was transferred to the cryo-transmission electron microscope for high-resolution imaging.