Cryo-transmission Electron Microscopy of Outer-inner Membrane Vesicles Naturally Secreted by Gram-negative Pathogenic Bacteria
Cryo-transmission Electron Microscopy of Outer-inner Membrane Vesicles Naturally Secreted by Gram-negative Pathogenic Bacteria
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DOI：
10.21769/bioprotoc.3367
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发表时间：
2019-09-20
期刊：
影响因子：
0.8
通讯作者：
Mercade, Elena
中科院分区：
文献类型：
--
作者：
Delgado, Lidia;Baeza, Nicolas;Mercade, Elena
开发了一种方案来可视化和分析来自革兰氏阴性细菌的膜囊泡（MV）的结构。现在人们普遍认为，这些微米球形囊泡通常由来自所有三个生命领域的细胞产生，因此该方案可用于研究真核生物和古细菌以及细菌产生的囊泡。MVs具有多种功能，涉及细胞通讯、免疫系统相互作用、发病机制和营养获取等，因此MVs成为研究的热点。由于MVs的尺寸较小（25-300 nm），需要使用电子显微镜观察MVs，通常通过负染MVs的透射电子显微镜（TEM）进行观察。用于其可视化的其他协议包括扫描电子显微镜，固定和包埋囊泡后的TEM，甚至原子力显微镜。在这些技术中的一些中，囊泡结构通过干燥而改变，而另一些是耗时的，并且它们中的大多数可以产生伪像。冷冻后的Cryo-TEM允许嵌入玻璃冰薄膜中的样品的可视化，这保留了它们的天然细胞结构并为成像提供了最高的可用分辨率。这是通过非常高的冷却速率来实现的，该冷却速率将细胞的固有水转变为玻璃状冰，从而避免晶体形成和水与溶质之间的相分离。除了其他类型的表征，MV结构的准确知识，这可以通过该协议获得，是必不可少的MV在不同领域的应用。
A protocol was developed to visualize and analyze the structure of membrane vesicles (MVs) from Gram-negative bacteria. It is now accepted that these micrometric spherical vesicles are commonly produced by cells from all three domains of life, so the protocol could be useful in the study of vesicles produced by eukaryotes and archaea as well as bacteria. The multiplicity of functions performed by MVs, related to cell communication, interaction with the immune system, pathogenesis, and nutrient acquisition, among others, has made MVs a hot topic of research.Due to their small size (25-300 nm), the observation of MVs requires electron microscopy and is usually performed by transmission electron microscopy (TEM) of negatively stained MVs. Other protocols applied for their visualization include scanning electron microscopy, TEM after fixation and embedding of vesicles, or even atomic force microscopy. In some of these techniques, vesicle structure is altered by drying, while others are time-consuming and most of them can generate artifacts. Cryo-TEM after plunge freezing allows the visualization of samples embedded in a thin film of vitreous ice, which preserves their native cellular structures and provides the highest available resolution for the imaging. This is achieved by very high cooling rates that turn the intrinsic water of cells into vitreous ice, avoiding crystal formation and phase segregation between water and solutes. In addition to other types of characterization, an accurate knowledge of MV structure, which can be obtained by this protocol, is essential for MV application in different fields.