title: 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 scrutinized. 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.
authors.affiliation: Department of Materials and London Centre for Nanotechnology, Imperial College London, Exhibition Road, London, SW7 2AZ, U.K.
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Materials and London Centre for Nanotechnology, Imperial College London, Exhibition Road, London, SW7 2AZ, U.K.
authors.forename: A E
authors.initials: AE
authors.lastname: Goode
authors.affiliation: Multi-Imaging Centre, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, U.K.
authors.forename: J N
authors.initials: JN
authors.lastname: Skepper
authors.affiliation: National Heart and Lung Institute, Imperial College London, SW3 6LY, U.K.
authors.forename: A J
authors.initials: AJ
authors.lastname: Thorley
authors.affiliation: National Heart and Lung Institute, Imperial College London, SW3 6LY, U.K.
authors.forename: J M
authors.initials: JM
authors.lastname: Seiffert
authors.affiliation: National Heart and Lung Institute, Imperial College London, SW3 6LY, U.K.
authors.forename: K F
authors.initials: KF
authors.lastname: Chung
authors.affiliation: National Heart and Lung Institute, Imperial College London, SW3 6LY, U.K.
authors.forename: T D
authors.initials: TD
authors.lastname: Tetley
authors.affiliation: Department of Chemistry and London Centre for Nanotechnology, Imperial College London, Exhibition Road, London, SW7 2AZ, U.K.
authors.forename: M S P
authors.initials: MS
authors.lastname: Shaffer
authors.affiliation: Department of Materials and London Centre for Nanotechnology, Imperial College London, Exhibition Road, London, SW7 2AZ, U.K.
authors.forename: M P
authors.initials: MP
authors.lastname: Ryan
authors.affiliation: Department of Materials and London Centre for Nanotechnology, Imperial College London, Exhibition Road, London, SW7 2AZ, U.K.
authors.forename: A E
authors.initials: AE
authors.lastname: Porter
mesh_terms: D016477:Artifacts; D020285:Cryoelectron Microscopy; D016591:Histocytological Preparation Techniques / Q000379:methods / Q000592:standards; D053768:Metal Nanoparticles / Q000648:ultrastructure*; D046529:Microscopy, Electron, Transmission; D015388:Organelles; D012834:Silver; D013194:Staining and Labeling; D013696:Temperature
chemical_list: D012834:Silver
keywords: Artefacts; biological sample preparation; electron microscopy; resin embedding; silver nanomaterials; staining; sulfidation; toxicity