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Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing numerous ILVs is called a multivesicular body (MVB). ILVs contain internalized molecules such as antigens, nucleic acids, proteins, and metabolites. Some of these molecules are released from the MVBs inside exosomes and are transported to other cells. Other MVBs contain molecules that are retained in the ILVs and are later degraded within the...
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Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis
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Method for Mid-IR Spectroscopy of Extracellular Vesicles at the Subvesicle Level.

Nikolaus Hondl1, Lena Neubauer1,2, Victoria Ramos-Garcia3

  • 1Institute of Chemical Technologies and Analytics, TU Wien, 1060 Vienna, Austria.

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|August 27, 2025
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Summary

This study introduces a new method using atomic force microscopy infrared spectroscopy (AFM-IR) to analyze the chemical makeup of individual extracellular vesicles (EVs). This technique offers nanoscale insights into EV structure and composition, crucial for understanding cell communication.

Keywords:
AFM-IRchemometricsextracellular vesiclesheterogeneityinfrared spectroscopynanoscale imaging

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Area of Science:

  • Biochemistry
  • Nanotechnology
  • Cell Biology

Background:

  • Extracellular vesicles (EVs) are key mediators of intercellular communication, transporting cellular components and influencing physiological and pathological processes.
  • Human milk EVs are implicated in the development of acquired immunity.
  • Current analytical methods lack the resolution for label-free chemical analysis at the single EV level.

Purpose of the Study:

  • To develop and present a protocol for nanoscale chemical imaging of individual EVs.
  • To enable label-free profiling of EV structure and composition at the single-vesicle level.

Main Methods:

  • Utilized atomic force microscopy infrared spectroscopy (AFM-IR), a nanoscale chemical imaging technique.
  • Developed a protocol involving EV immobilization on anti-CD9 antibody-functionalized silicon surfaces via microcontact printing.
  • Employed hyperspectral imaging combined with image processing for drift correction and individual vesicle selection to analyze numerous EVs.

Main Results:

  • AFM-IR successfully provided size information and mid-infrared spectra of individual EVs with sub-vesicle spatial resolution.
  • The spectral data obtained from individual EVs showed favorable comparison with bulk reference spectra.
  • The protocol demonstrated the capability to acquire spectral information from a large number of EVs.

Conclusions:

  • The presented AFM-IR protocol offers a powerful tool for detailed structural and chemical characterization of individual EVs.
  • This method advances the understanding of EV heterogeneity and function, particularly in contexts like human milk and immunity.
  • The technique overcomes limitations of current methods, enabling label-free, high-resolution analysis of nanoscale biological particles.