Chemical characterization of extracellular vesicles at the sub-vesicle level

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.