Extracellular vesicles (EVs) are nanosized particles enclosed by a lipid bilayer that are released by cells, which are associated with various physiological and pathological functions. EVs are present in all biological fluids and they contain specific cellular components such as lipids, proteins, nucleic acids, RNAs and metabolites. [1, 2] In human milk EVs are believed to be important for the nutrition of infants and for the development of their acquired immunity. State-of-the-art analysis methods are not able to provide label free chemical information at the single vesicle level, hence new analysis techniques are required to study the chemical difference withing EV (sub-)population. [3] Already existing protocols for AFM-IR do not provide this either. Therefore we introduce a protocol to profile structure and composition of individual EVs beyond the analysis of earlier protocols. [4] First, EVs are selectively immobilized onto a silicone surface using microcontact printed Anti-CD9 antibodies. AFM-IR tapping mode then provides both EV size and mid-infrared spectra of the fragile individual vesicles. In addition, AFM-IR images provide spectral information for multiple EVs within a single measurement. Stacking images taken at different wavenumbers yields hyperspectral chemical images of vesicles at nanoscale spatial resolution. Chemometrics is used to break down the chemical composition of the individual EVs. In this work we demonstrated chemical analysis of individual extracellular vesicles with our new established protocol. Nanoscale AFM-IR spectra of EVs compare favourably to bulk reference spectra. Principal component analysis (PCA) was applied to find heterogeneity of composition within individual. Due to the EV size information provided by AFM, it is possible to study correlations between vesicle size and their chemical composition. References [1] Hessvik, N.P., et al. ”Current knowledge on exosome biogenesis and release.” Cell. Mol. Life Sci. 75, 193–208 (2018). [2] Daniella Schatz, et al. “Extracellular vesicles — new players in cell–cell communication in aquatic environments” Current Opinion in Microbiology, Volume 43 148-154 (2018) [3] Théry, Clotilde, et al. "Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines." Journal of extracellular vesicles 7.1 (2018) [4] Kim, S. Y., et al. “None of us is the same as all of us: resolving the heterogeneity of extracellular vesicles using single-vesicle, nanoscale characterization with resonance enhanced atomic force microscope infrared spectroscopy (AFM-IR). “, Nanoscale Horizons, (2018)