Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals.
Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals.
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DOI：
10.1016/j.micron.2017.03.019
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发表时间：
2017-08
期刊：
影响因子：
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通讯作者：
Lucocq J
中科院分区：
文献类型：
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作者：
Asadi J;Ferguson S;Raja H;Hacker C;Marius P;Ward R;Pliotas C;Naismith J;Lucocq J
醋酸铀酰/钨双染剂被提出用于在TEM中成像富含脂质的纳米颗粒。结合甲基纤维素包埋，该技术提高了膜的对比度。该技术适用于脂质体、纳米盘和双胞。双重染色应改善富含脂质的纳米颗粒的定量。合成和天然存在的富脂质纳米颗粒在生物医学中具有广泛的重要性。它们包括脂质体、双胞体、纳米盘、外来体和病毒颗粒。对这些颗粒的定量研究需要对整个种群进行高分辨率可视化的方法。一种强大的成像方法是玻璃化样品的冷冻-EM，但这在技术上要求很高，需要专门的设备，提供低对比度，并且不能揭示群体中存在的所有颗粒。另一种方法是经典的负染色-EM，其更容易获得，但难以标准化用于更大的造影结构，其易于产生结构塌陷和对比度变化的伪影。第三种方法是用醋酸双氧铀作为对比剂包埋在甲基纤维素薄膜中。甲基纤维素包埋已被广泛用于对比和支持冷冻切片，但仅偶尔用于可视化富含脂质的囊泡结构，如内体和外泌体。在这里，我们提出了一个简单的甲基纤维素为基础的方法，常规和全面的可视化合成的脂质丰富的纳米颗粒制剂，如脂质体，bicelles和nanodisks。它结合了醋酸铀酰（UA）和钨基电子染料（即磷钨酸（PTA）或硅钨酸钠（STA））与甲基纤维素包埋的新型双染色混合物。虽然甲基纤维素在干燥期间支持精细的脂质结构，但与单独的UA相比，向UA中添加PTA或STA提供了脂质结构显示和对比度的显著增强。这种双重染色方法应有助于常规结构评价和富脂质纳米颗粒结构的定量。
Uranyl acetate/tungsten double stains are proposed for imaging lipid rich nanoparticle in TEM. Combined with methylcellulose embedment, the technique enhances membrane contrast. The technique works for liposomes, nanodiscs and bicelles. The double staining should improve quantification of lipid rich nanoparticles. 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.