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Reference: Asadi, J, Ferguson, S, Raja, H et al., (2017). Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals. Micron, 99, 40-48.Citable link to this page:

 

Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals

Abstract: Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain- EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

Publication status:PublishedPeer Review status:Peer reviewedVersion:Publisher's versionDate of acceptance:2017-03-29 Funder: University of St Andrews   Funder: Scottish Government   Funder: Tenovus Scotland   Funder: Wellcome Trust   Notes:© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

Bibliographic Details

Publisher: Elsevier

Publisher Website: http://www.elsevier.com/

Journal: Micronsee more from them

Publication Website: http://www.sciencedirect.com/science/journal/09684328

Volume: 99

Issue Date: 2017-04

pages:40-48Identifiers

Doi: https://doi.org/10.1016/j.micron.2017.03.019

Issn: 0968-4328

Eissn: 1878-4291

Uuid: uuid:5eb7121a-30d5-428a-a875-628f30cdf166

Urn: uri:5eb7121a-30d5-428a-a875-628f30cdf166

Pubs-id: pubs:700412 Item Description

Type: journal-article;

Version: Publisher's versionKeywords: Bicelles Phospholipids Membranes Lipids Methylcellulose Uranyl acetate Phosphotungstic acid Sodium silicotungstate Negative stain Liposomes Nanodiscs

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Author: Asadi, J - - - Ferguson, S - - - Raja, H - - - Hacker, C - - - Marius, P - - - Ward, R - - - Pliotas, C - - - Naismith, J - Oxfor

Source: https://ora.ox.ac.uk/objects/uuid:5eb7121a-30d5-428a-a875-628f30cdf166



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