Low dose analysis of nanoparticles suspended in vitreous ice for near native state imaging
Authors: Hondow Nicole , Ward Michael , Brydson Rik , Brown Andy 
Published: December 2016

Most formulated fine chemical products are complex systems that contain multiple components, with nanoparticles and any incorporated surface coatings interacting with other particles and the dispersant (which can be a liquid or solid). Assessing nanoparticles when suspended in a liquid can be challenging as the particles may disperse individually, agglomerate, aggregate, sediment, chemically‐alter or even dissolve and re‐precipitate. With the appropriate sample preparation however, TEM can be used to measure the dispersion and any transformation of nanoparticles suspended in, for example, biological or environmental media [1,2].
Conventional transmission electron microscopy (TEM), with samples prepared by simply drop‐casting suspensions onto a thin carbon film, enables imaging and analysis of individual nanoparticles but, because of the drying process, does not capture the particle agglomeration in the dispersion or the surface chemistry when hydrated [3]. To overcome this problem we have prepared thin sections of nanoparticle suspensions for TEM by plunge‐freezing a blotted grid into liquid ethane to ensure the aqueous phase vitrifies with no significant redistribution of suspended material. We have used this technique to quantify the dispersion of polymer coated quantum dots, silica and zinc oxide nanoparticles in water and biological cell culture media, identifying the true form in which these nanoparticles are taken up into cells in vitro and thereby providing mechanistic insight to the cellular response at these exposures [3,4,5].

Low dose electron microscopy of nanoparticles suspended in vitreous ice provides opportunity for the analysis of the structure
and
chemistry of the dispersion, both vital characteristics to understand before any successful biomedical exploitation of nanoparticles. Here, dextran coated iron oxide nanoparticles agglomerated in aqueous suspension and captured in vitreous ice were imaged by bright field TEM and analysed by energy dispersive X‐ray (EDX) spectroscopy. Careful control of the illumination conditions (electron dose) permit near native state imaging and confirmation of composition before inducing significant damage to the surrounding ice matrix and subsequent movement of the particles (Figure 1). HAADF STEM imaging was conducted using a 1.3 Å probe and 60 pA probe current, with a resulting EDX map collected in just over one minute showing an iron signal appropriately localised to the nanoparticles (Figure 2).

Going forward, we will use the recently installed FEI Titan Cubed Themis 300 G2 S/TEM at the University of Leeds which is equipped with FEI SuperX EDX spectrometers, a Gatan Quantum ER imaging filter and Gatan OneView CCD to explore the limits of nanoparticle structural analysis (incorporating diffraction and lattice imaging), as well as use of STEM‐EDX and electron energy loss spectroscopy for detailed elemental analysis when encased in vitreous ice. In addition to examining the dispersion state of nanoparticles in different suspensions, our goal is to identify and analyse the surface coatings on nanoparticles in the frozen hydrated state, thereby extending the capability of near native state imaging and analysis of nanoparticle suspensions by TEM.