Imaging of Extracellular Vesicles by Atomic Force Microscopy.

Area of Science:

• Biophysics
• Nanotechnology
• Cell Biology

Background:

• Extracellular vesicles (EVs), including exosomes, are crucial for intercellular communication.
• Accurate characterization of EV size is essential but challenging with traditional methods like cryo-transmission electron microscopy (cryo-TEM).
• Cryo-TEM is limited by cost, expertise, and sample size, necessitating alternative techniques.

Purpose of the Study:

• To present a protocol for utilizing Atomic Force Microscopy (AFM) for extracellular vesicle (EV) size characterization.
• To demonstrate AFM as a viable and accessible alternative to cryo-TEM for EV sizing.
• To validate AFM-based EV sizing against established cryo-TEM methods.

Main Methods:

• Developed a protocol for sample preparation, electrostatic immobilization, and imaging of EVs using AFM.
• Acquired 3D topographical data of EVs on a modified mica substrate.
• Analyzed AFM data to determine EV size and biophysical properties.

Main Results:

• AFM imaging allows for predictable and customizable fixation of EVs on a modified mica surface.
• The protocol enables the analysis of a large number of EVs, overcoming limitations of cryo-TEM.
• EV sizing results obtained via AFM are consistent with those from cryo-TEM imaging.

Conclusions:

• AFM provides a versatile, accessible, and scalable method for characterizing the size and biophysical properties of extracellular vesicles.
• This AFM-based protocol offers a practical alternative for researchers needing to analyze large populations of EVs.
• The consistency of AFM results with cryo-TEM validates its utility in EV research.