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Revealing Sources of Variation for Reproducible Imaging of Protein Assemblies by Electron Microscopy

1
National Physical Laboratory, Teddington, Middlesex TW11 0LW, UK
2
CEM Corporation, Buckingham Industrial Park, Buckingham MK18 1WA, UK
3
Biomedical Imaging Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK
*
Author to whom correspondence should be addressed.
Micromachines 2020, 11(3), 251; https://doi.org/10.3390/mi11030251
Received: 21 January 2020 / Revised: 24 February 2020 / Accepted: 26 February 2020 / Published: 27 February 2020
(This article belongs to the Special Issue Self-Assembly of Microcomponents)
Electron microscopy plays an important role in the analysis of functional nano-to-microstructures. Substrates and staining procedures present common sources of variation for the analysis. However, systematic investigations on the impact of these sources on data interpretation are lacking. Here we pinpoint key determinants associated with reproducibility issues in the imaging of archetypal protein assemblies, protein shells, and filaments. The effect of staining on the morphological characteristics of the assemblies was assessed to reveal differential features for anisotropic (filaments) and isotropic (shells) forms. Commercial substrates and coatings under the same staining conditions gave comparable results for the same model assembly, while highlighting intrinsic sample variations including the density and heterogenous distribution of assemblies on the substrate surface. With no aberrant or disrupted structures observed, and putative artefacts limited to substrate-associated markings, the study emphasizes that reproducible imaging must correlate with an optimal combination of substrate stability, stain homogeneity, accelerating voltage, and magnification. View Full-Text
Keywords: protein self-assembly; protein filaments; virus-like capsids; electron microscopy; negative staining protein self-assembly; protein filaments; virus-like capsids; electron microscopy; negative staining
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MDPI and ACS Style

Kepiro, I.E.; Nardone, B.; Page, A.; Ryadnov, M.G. Revealing Sources of Variation for Reproducible Imaging of Protein Assemblies by Electron Microscopy. Micromachines 2020, 11, 251.

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