Using liquid cell TEM to understand and characterise complex hydrated systems
DOI: 10.22443/rms.mmc2025.185
Authors
Martha Ilett (1), Zabeada Aslam (1), Stuart Micklethwaite (1), Andy Brown (1), Rik Drummond-Brydson (1), Nicole Hondow (1)
Affiliations
- University of Leeds
In this work, we use liquid cell transmission electron microscopy (LCTEM) to try and understand the dispersion of nanoparticles in biological media, alongside understanding and overcoming some of the complexities of LCTEM analysis through a combination of experimental and theoretical studies.
Today nanoparticles are used in a huge range of nanotechnological applications, including within medicine, construction and consumer products. In many cases they are required to be dispersed in liquids for these applications e.g. for drug delivery[1]. This will inherently change their properties and accordingly their functionality[2, 3]. However, studying these changes can be difficult using standard TEM approaches since the high vacuum environment requires samples to be dry for analysis. As such the use of advanced in situ techniques which allow nanoparticles to be studied in solution must be used. One such technique is LCTEM which permits a liquid sample to be sealed from the vacuum and high-resolution characterisation of a hydrated nanoparticle suspension to be achieved. However, LCTEM itself is a complicated technique influenced heavily by beam-liquid interactions that still need to be fully understood[4, 5]. In this work we use LCTEM technologies to try and illicit a better understanding of nanoparticles when suspended in biological media and through this aim to better understand the complexities of LCTEM and develop workflows to overcome some of these limitations.
A number of nanoparticles (ceria, silica, gold) were dispersed in a range of biological fluids including Dulbecco’s modified eagle’s medium (DMEM), DMEM plus fetal bovine serum (FBS) and phosphate buffered saline (PBS). Initial bulk analysis was carried out via dynamic light scattering to understand any changes in size and/or dispersion. Following this a Hummingbird Scientific liquid cell TEM holder was used to carry out TEM imaging in situ. An FEI Titan3 Themis G2 operating at 300 kV and equipped with a Gatan OneView CCD was used for all TEM analysis. Alongside experimental studies COMSOL was used to model the fluid dynamics within the liquid cell holder and understand the flow of liquids over the SiN window.
We found that the dispersion state of ceria nanoparticles suspended in DMEM with and without 10% FBS dramatically changes. Without the presence of FBS the ceria nanoparticles heavily agglomerate and drop out of solution, whilst in comparison the addition of FBS stabilises the ceria particles in solution. This was shown using LCTEM where real time images of the nanoparticles in solution were captured. Concurrently we found that loading the cell wet results in more nanoparticles within the window whereas loading empty and then flowing through the solution leads to complications whereby most nanoparticles are pushed to the surrounding regions of the chip rather than across the SiN window. This was further understood through fluid dynamic simulations using COMSOL which showed preferential flow of liquids around the window. Furthermore, electron beam induced radiolysis is known to cause changes in the pH of the solution during LCTEM analysis. This was shown here by monitoring ceria nanoparticle dissolution and was utilised to try and mimic some of the pH changes that are known to occur during entry of nanoparticles into the cell via endocytosis.
Overall, we show that LCTEM can be used to study nanoparticle dispersions in biological media and in addition suggest experimental workflows that might overcome some of the complexities of LCTEM techniques. These will have far reaching benefits within many fields of scientific research allowing high resolution characterisation of more complex hydrated systems.
References
1. Mitchell, M.J., et al., Engineering precision nanoparticles for drug delivery. Nature Reviews Drug Discovery, 2021. 20(2): p. 101-124.
2. Ilett, M., et al., Nanoparticle corona artefacts derived from specimen preparation of particle suspensions.Scientific Reports, 2020. 10(1): p. 5278.
3. Mahmoudi, M., et al., The protein corona from nanomedicine to environmental science. Nature Reviews Materials, 2023. 8(7): p. 422-438.
4. Pu, S., C. Gong, and A.W. Robertson, Liquid cell transmission electron microscopy and its applications. Royal Society Open Science, 2020. 7(1): p. 191204.
5. Schneider, N.M., et al., Electron–Water Interactions and Implications for Liquid Cell Electron Microscopy. The Journal of Physical Chemistry C, 2014. 118(38): p. 22373-22382.
Keywords
Liquid Cell TEM
Nanoparticles
in situ TEM