Brownian Motion Influence on AFM Exosomes' Size Measurements

Area of Science:

• Biophysics
• Nanotechnology
• Cell Biology

Background:

• Extracellular vesicles (EVs) are crucial for intercellular communication.
• Accurate size characterization of EVs is vital for understanding their function.
• Nanoparticle tracking analysis (NTA) measures hydrodynamic diameter, while atomic force microscopy (AFM) measures geometric diameter.

Purpose of the Study:

• To investigate the impact of Brownian motion and sample preparation time on AFM-based EV size measurements.
• To establish the relationship between geometric (AFM) and hydrodynamic (NTA) sizes of EVs.
• To compare experimental findings with Monte Carlo simulations for exosome analysis.

Main Methods:

• Evaluation of exosomes from the PC3 human prostate cancer cell line using NTA and AFM.
• Analysis of size measurements under varying sample drop conditions and preparation times.
• Application of Monte Carlo simulations to model and validate experimental results.

Main Results:

• AFM measurements of exosome size on mica substrates are initially underestimated due to Brownian motion and lipid bilayer stretching.
• Thinner sample drops and shorter preparation times lead to faster and more accurate AFM size determination.
• A linear scaling factor connects the geometric (AFM) and hydrodynamic (NTA) diameters, characterizing the EVs.

Conclusions:

• Brownian motion and lipid bilayer dynamics significantly affect AFM imaging of EVs.
• Optimized sample preparation is critical for reliable AFM-based exosome size quantification.
• The identified scaling factor provides a method to correlate AFM and NTA size data for EV characterization.