Evaluation of correlated studies using liquid cell and cryo-transmission electron microscopy: Hydration of calcium sulphate and the phase transformation pathways of bassanite to gypsum.

Insight into the nucleation, growth and phase transformations of calcium sulphate could improve the performance of construction materials, reduce scaling in industrial processes and aid understanding of its formation in the natural environment. Recent studies have suggested that the calcium sulphate pseudo polymorph, gypsum (CaSO4 ·2H2 O) can form in aqueous solution via a bassanite (CaSO4 ·0.5H2 O) intermediate. Some in situ experimental work has also suggested that the transformation of bassanite to gypsum can occur through an oriented assembly mechanism. In this work, we have exploited liquid cell transmission electron microscopy (LCTEM) to study the transformation of bassanite to gypsum in an undersaturated aqueous solution of calcium sulphate. This was benchmarked against cryogenic TEM (cryo-TEM) studies to validate internally the data obtained from the two microscopy techniques. When coupled with Raman spectroscopy, the real-time data generated by LCTEM, and structural data obtained from cryo-TEM show that bassanite can transform to gypsum via more than one pathway, the predominant one being dissolution/reprecipitation. Comparisons between LCTEM and cryo-TEM also show that the transformation is slower within the confined region of the liquid cell as compared to a bulk solution. This work highlights the important role of a correlated microscopy approach for the study of dynamic processes such as crystallisation from solution if we are to extract true mechanistic understanding.

Application of automated electron microscopy imaging and machine learning to characterise and quantify nanoparticle dispersion in aqueous media.

For many nanoparticle applications it is important to understand dispersion in liquids. For nanomedicinal and nanotoxicological research this is complicated by the often complex nature of the biological dispersant and ultimately this leads to severe limitations in the analysis of the nanoparticle dispersion by light scattering techniques. Here we present an alternative analysis and associated workflow which utilises electron microscopy. The need to collect large, statistically relevant datasets by imaging vacuum dried, plunge frozen aliquots of suspension was accomplished by developing an automated STEM imaging protocol implemented in an SEM fitted with a transmission detector. Automated analysis of images of agglomerates was achieved by machine learning using two free open-source software tools: CellProfiler and ilastik. The specific results and overall workflow described enable accurate nanoparticle agglomerate analysis of particles suspended in aqueous media containing other potential confounding components such as salts, vitamins and proteins. LAY DESCRIPTION: In order to further advance studies in both nanomedicine and nanotoxicology, we need to continue to understand the dispersion of nanoparticles in biological fluids. These biological environments often contain a number of components such as salts, vitamins and proteins which can lead to difficulties when using traditional techniques to monitor dispersion. Here we present an alternative analysis which utilises electron microscopy. In order to use this approach statistically relevant large image datasets were collected from appropriately prepared samples of nanoparticle suspensions by implementing an automated imaging protocol. Automated analysis of these images was achieved through machine learning using two readily accessible freeware; CellProfiler and ilastik. The workflow presented enables accurate nanoparticle dispersion analysis of particles suspended in more complex biological media.