Abstract：
Bacterial extracellular vesicles (BEVs) have emerged as mediators of transkingdom communication with numerous potential biotechnological applications. As such, investigation of BEV\'s protein composition holds promise to uncover new biological mechanisms, such as in microbiome-host communication or pathogen infection. Additionally, bioengineering of BEV protein composition can enhance their therapeutic potential. However, accurate assessment of BEV protein cargo is limited by their nanometer size, which precludes light microscopy imaging, as well as by co-isolation of protein impurities during separation processes. A solution to these challenges is found in immunogold transmission electron microscopy (TEM), which combines antibody-based labeling with direct visualization of BEVs. Several challenges are commonly encountered during immunogold TEM analysis of BEVs, most notably inefficient antibody labeling and poor contrast. Here, we present an optimized protocol for immunogold TEM analysis of BEVs that overcomes such challenges.
摘要：
细菌细胞外囊泡（BEV）已成为具有许多潜在生物技术应用的跨王国通讯的介体。因此，对BEV蛋白质组成的研究有望发现新的生物学机制，例如在微生物组-宿主通讯或病原体感染中。此外,BEV蛋白质组成的生物工程可以增强其治疗潜力。然而,BEV蛋白质货物的准确评估受到其纳米尺寸的限制，排除了光学显微镜成像，以及在分离过程中共同分离蛋白质杂质。在免疫金透射电子显微镜（TEM）中找到了解决这些挑战的方法，它将基于抗体的标记与BEV的直接可视化相结合。在BEV的免疫金TEM分析过程中通常会遇到几个挑战，最值得注意的是无效的抗体标记和差的对比度。这里,我们提出了一种针对BEV的免疫金TEM分析的优化方案，该方案克服了这些挑战。
