Laboratoire Matière et Systèmes Complexes, UMR7057 CNRS/Université Paris Diderot, Paris, France.
Nanoscale. 2018 Jan 18;10(3):1234-1244. doi: 10.1039/c7nr07576f.
Cell-derived extracellular vesicles (EVs) circulating in body fluids hold promises as bioactive therapeutic agents and as biomarkers to diagnose a wide range of diseases. However nano-imaging methods are needed to characterize these complex and heterogeneous soft materials in their native wet environment. Herein, we exploit liquid-cell transmission electron microscopy (LCTEM) to characterize the morphology and dynamic behavior of EVs in physiological media with nanometer resolution. The beam-induced controlled growth of Au nanoparticles on bilayer membranes is used as an original in situ staining method to improve the contrast of EVs and artificial liposomes. LCTEM provides information about the size distribution and concentration of EVs that are consistent with Cryo-TEM and nanoparticle tracking analysis measurements. Moreover, LCTEM gives a unique insight into the dynamics of EVs depending on their liquid environment. The size-dependent morphology of EVs is sensitive to osmotic stress which tends to transform their spherical shape to ellipsoidal, stomatocyte or discocyte morphologies. In the liquid-cell, EVs exhibit a sub-diffusive motion due to strong interactions between the Au nanoparticles and the liquid-cell windows. Finally, the high-resolution monitoring of EV aggregation and fusion illustrate that LCTEM opens up a new way to study cell-membrane dynamics.
细胞衍生的细胞外囊泡 (EVs) 在体液中循环,有望成为生物活性治疗剂和诊断广泛疾病的生物标志物。然而,需要纳米成像方法来表征这些复杂且异质的软物质在其自然湿环境中的特性。在此,我们利用液室透射电子显微镜 (LCTEM) 以纳米分辨率在生理介质中对 EVs 的形态和动态行为进行表征。利用金纳米颗粒在双层膜上的束诱导可控生长作为原始的原位染色方法,以提高 EVs 和人工脂质体的对比度。LCTEM 提供了有关 EVs 的尺寸分布和浓度的信息,这些信息与 Cryo-TEM 和纳米颗粒跟踪分析测量结果一致。此外,LCTEM 还深入了解了 EVs 的动力学特性,这取决于它们的液体环境。EVs 的尺寸依赖性形态对渗透压敏感,倾向于将其球形转变为椭圆形、孔板状或圆盘状形态。在液室中,由于 Au 纳米颗粒与液室窗之间的强烈相互作用,EVs 表现出亚扩散运动。最后,对 EV 聚集和融合的高分辨率监测表明,LCTEM 为研究细胞膜动力学开辟了新途径。