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

Asadi, Jalal; Ferguson, Sophie; Raja, Haseem; Hacker, Christian; Marius, Phedra; Ward, Roger; Pliotas, Christos; Naismith, James H.; Lucocq, John M.

doi:10.1016/j.micron.2017.03.019

Cited by 36 publications

(28 citation statements)

References 40 publications

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“…As we previously observed with nanodiscs [ 36 ], thin films of mixed metal stain MC reduced the profile asymmetry so that the mean major–minor axes measurement ratio of adsorbed plasma LPs was 1.11 (SD = 0.096, n = 32), as compared to an expected ratio for LDL of 1.77 (major axis 21.4 ± 1.3 nm and minor axis 12.1 ± 1.1 nm [ 45 ]). This was consistent with the particles lying flat and presenting en face to the electron beam (note that this effect might not be relevant for larger, less asymmetric but angular VLDL particles [ 41 ]).…”

Section: Resultssupporting

confidence: 61%

“…Enhancement of edge contrast was observed after including sodium silicotungstate (STA) in the staining mixture ( Figure 1 b, mixed metal staining). This modification has previously been shown to enhance contrast on a range of nanoparticles such as lipid-rich nanodiscs [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

“…An additional advantage of thinner MC films is that flattened spheroidal particles such as LDL and nanodiscs tend to orient en face to the film, rather than taking up a range of orientations [ 34 , 36 , 37 ]. This effect makes the particles present a more homogeneous-sized profile to the electron beam.…”

Section: Resultsmentioning

confidence: 99%

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Developing Electron Microscopy Tools for Profiling Plasma Lipoproteins Using Methyl Cellulose Embedment, Machine Learning and Immunodetection of Apolipoprotein B and Apolipoprotein(a)

Giesecke

Soete

Mackinnon

et al. 2020

IJMS

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Plasma lipoproteins are important carriers of cholesterol and have been linked strongly to cardiovascular disease (CVD). Our study aimed to achieve fine-grained measurements of lipoprotein subpopulations such as low-density lipoprotein (LDL), lipoprotein(a) (Lp(a), or remnant lipoproteins (RLP) using electron microscopy combined with machine learning tools from microliter samples of human plasma. In the reported method, lipoproteins were absorbed onto electron microscopy (EM) support films from diluted plasma and embedded in thin films of methyl cellulose (MC) containing mixed metal stains, providing intense edge contrast. The results show that LPs have a continuous frequency distribution of sizes, extending from LDL (> 15 nm) to intermediate density lipoprotein (IDL) and very low-density lipoproteins (VLDL). Furthermore, mixed metal staining produces striking “positive” contrast of specific antibodies attached to lipoproteins providing quantitative data on apolipoprotein(a)-positive Lp(a) or apolipoprotein B (ApoB)-positive particles. To enable automatic particle characterization, we also demonstrated efficient segmentation of lipoprotein particles using deep learning software characterized by a Mask Region-based Convolutional Neural Networks (R-CNN) architecture with transfer learning. In future, EM and machine learning could be combined with microarray deposition and automated imaging for higher throughput quantitation of lipoproteins associated with CVD risk.

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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

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Developing Electron Microscopy Tools for Profiling Plasma Lipoproteins Using Methyl Cellulose Embedment, Machine Learning and Immunodetection of Apolipoprotein B and Apolipoprotein(a)

Giesecke

Soete

Mackinnon

et al. 2020

IJMS

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“…Other encapsulation techniques, such as electrospraying, are known to obtain a narrow particle size distribution; this is not always the case with IG. Polydispersion indices (PDI) are usually higher than 0.1, reaching values up to 0.5 .…”

Section: Disadvantagesmentioning

confidence: 99%

Ionotropic gelation method in the synthesis of nanoparticles/microparticles for biomedical purposes

Pedroso‐Santana

Fleitas‐Salazar

2020

Polymer International

168

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The encapsulation of drugs inside polymeric nanoparticles/microparticles is a strategy currently employed in the search for new and more effective therapies. The use of biocompatible and biodegradable polymers gives several advantages to these formulations. Protection of the active principals against the action of environmental and physiological agents, the reduced number of doses and a subsequent decrease in drug-related adverse effects, and increased bioavailability are some of these advantages. Several methods and materials are now used to synthesize nanoparticles/microparticles for biomedical applications, from carbon-derived structures to metallic and lipid particles. However, among the methods using polymers, ionotropic gelation is one of the more affordable and easier procedures to perform in daily laboratory work. In this mini-review, we address relevant characteristics of ionotropic gelation, beginning with basic aspects of the technique, which reagents and conditions are commonly used, the factors affecting the stability of the formulation, the advantages and disadvantages of the method and some principal characterization techniques for the nanoparticulate/microparticulate formulation. Finally, we conclude with a few important considerations.

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“…Samples were prepared for transmission electron microscopy as described in Asadi et al 22 Briefly, 0.5 mg ml À1 SUVs made from soybean polar lipids, DMPC or POPC, in the presence and absence of SMA copolymer, were adsorbed on 200 mesh formvar coated copper grids for five minutes followed by washing on 6 drops with milliQ H 2 O. Grids were then incubated for five minutes on ice on drops with methylcellulose-uranylacetate mixture (800 ml 2% methylcellulose and 200 ml 4% uranyl acetate), looped out and dried. Images were recorded at 120 kV on a JEOL 1400plus Transmission Electron Microscope.…”

Section: Transmission Electron Microscopy (Tem) Experimentsmentioning

confidence: 99%

Styrene maleic acid copolymer induces pores in biomembranes

et al. 2019

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

Cited by 36 publications

References 40 publications

Developing Electron Microscopy Tools for Profiling Plasma Lipoproteins Using Methyl Cellulose Embedment, Machine Learning and Immunodetection of Apolipoprotein B and Apolipoprotein(a)

Developing Electron Microscopy Tools for Profiling Plasma Lipoproteins Using Methyl Cellulose Embedment, Machine Learning and Immunodetection of Apolipoprotein B and Apolipoprotein(a)

Ionotropic gelation method in the synthesis of nanoparticles/microparticles for biomedical purposes

Styrene maleic acid copolymer induces pores in biomembranes

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