Faculty of Medicine and Health, Sydney Pharmacy School, The University of Sydney, Sydney, NSW, Australia.
Sydney Nano Institute, The University of Sydney, Sydney, NSW, Australia.
Nat Protoc. 2019 Feb;14(2):576-593. doi: 10.1038/s41596-018-0109-3.
Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.
细胞外囊泡 (EVs) 是高度专业化的纳米级组装体，可传递复杂的生物有效负载，以介导细胞间通讯。EVs 具有异质性，对这种异质性进行特征描述对于理解 EV 的生物发生和活性以及将其与生物反应和病理联系起来至关重要。研究 EV 组成的传统方法通常缺乏分辨率和/或灵敏度来表征单个 EV，因此评估 EV 的异质性仍然具有挑战性。我们最近开发了一种原子力显微镜红外光谱 (AFM-IR) 方法，以纳米级分辨率探测单个 EV 的结构组成。在这里，我们提供了我们方法的分步程序，并展示了其揭示单个 EV 内、同一 EV 群体内和不同 EV 群体之间异质性的能力。我们的方法是无标记的，能够在单个 EV 内检测脂质、蛋白质和核酸。从细胞培养基中分离 EV 后，该方案涉及将 EV 样品孵育在合适的基底上、设置 AFM-IR 仪器并收集纳米红外光谱和纳米红外图像。数据采集和分析可以在 24 小时内完成，并且只需要对光谱学和化学有基本的了解。我们预计，通过 AFM-IR 对 EV 组成和结构的新认识将有助于我们对 EV 生物学的生物学理解，并可能在疾病诊断和 EV 治疗的开发中找到应用。
