Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

Abstract

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinised. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

Silver nanoparticles reduce brain inflammation and related neurotoxicity through induction of H2S-synthesizing enzymes

“…Ultrathin cell monolayers (approximately 50–100 nm) were cut with a 35° diamond knife and each section was examined in a JEOL 2000 transmission electron microscope at an accelerating voltage of 80 kV. The use of heavy metal stains to enhance cell ultrastructure in the TEM can alter the morphology and accelerate the oxidation of AgNPs 72 , and therefore was avoided. Our previous published work has shown that the sample preparation procedure applied in this study does not alter the physicochemistry of the AgNPs in the cellular environment 72 .…”

Corrosion processes of triangular silver nanoparticles compared to bulk silver

“…The accelerated ion release when defects intersect the surface can also explain the accelerated corrosion previously observed at the tips of nanorods, as these particles can have a multi-twinned internal structure with defects running along the length of the rod and intersecting at the surface of the tip (Chen et al 2015;Chen et al 2013a;Elechiguerra et al 2005).…”

Interactions and effects of metal oxide nanoparticles on microorganisms involved in biological wastewater treatment

“…The organic matter and nitrogen removal occur as biological processes developed by activated sludge, which can be altered by NPs presence. Chen et al () studied the functional bacteria of activated sludge in the presence of ZnO NPs. They showed remarkable changes in biodiversity even when nitrogen removal is slightly affected by ZnO NPs, as is our case.…”

“…The misinterpretation of imaging is incorrect due to some undesirable artefacts must be carefully considered. These artefacts are related to electron‐dense material present in the raw wastewater, metal ions associated with some microorganisms, or chemical compounds formed through sample preparation process (Chen et al, ). Therefore, TEM imaging should be complemented with analytical or diffraction techniques, to verify the chemical composition of NPs interacting with microorganisms.…”