Extracellular vesicles (EVs) are nanosized particles, which are associated with various physiological and pathological functions. They play a key role in inter-cell communication and are used as transport vehicles for various cell components. In human milk, EVs are believed to be important for the development of acquired immunity. State-of-the-art analysis methods are not able to provide label free chemical information at the single-vesicle level. We introduce a protocol to profile structure and composition of individual EVs with the help of photothermal scanning probe infrared spectroscopy (AFM-IR), a nanoscale chemical imaging technique. The protocol includes immobilization of EVs onto a silicon surface functionalized with anti-CD9 antibodies via microcontact printing. AFM-IR measurements of immobilized EVs provide size information and mid-infrared spectra at sub-vesicle spatial resolution. The received spectra compare favorably to bulk reference spectra. A key part of our protocol is a technique to acquire spectral information on a large number of EVs through hyperspectral imaging combined with image processing to correct for image drifts and selecting individual vesicles.

中文翻译：

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亚囊泡水平细胞外囊泡的化学表征


细胞外囊泡 （EV） 是纳米级颗粒，与各种生理和病理功能有关。它们在电池间通信中起着关键作用，并用作各种电池组件的运输工具。在母乳中，EV 被认为对获得性免疫力的发展很重要。最先进的分析方法无法在单囊泡水平上提供无标记的化学信息。我们介绍了一种协议，在光热扫描探针红外光谱 （AFM-IR）（一种纳米级化学成像技术）的帮助下分析单个 EV 的结构和组成。该方案包括通过微接触打印将 EV 固定在用抗 CD9 抗体功能化的硅表面上。固定化 EV 的 AFM-IR 测量以亚囊泡空间分辨率提供尺寸信息和中红外光谱。接收到的光谱优于块参考光谱。我们协议的一个关键部分是一种技术，通过高光谱成像结合图像处理来获取大量 EV 的光谱信息，以校正图像漂移和选择单个囊泡。