Method for Mid-IR Spectroscopy of Extracellular Vesicles at the Subvesicle Level

Extracellular vesicles (EVs) are nanosized particles that are associated with various physiological and pathological functions. They play a key role in intercell 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 the structure and composition of individual EVs with the help of atomic force microscopy infrared spectroscopy (AFM-IR), a nanoscale chemical imaging technique. The protocol includes the 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 subvesicle spatial resolution. The received spectra compare favorably to bulk reference spectra. A key part of our protocol is a technique to acquire spectral information about a large number of EVs through hyperspectral imaging combined with image processing to correct for image drift and select individual vesicles.