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Related Experiment Video

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Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy
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An Optimized Positive Staining Protocol Method for Clear Visualization of Extracellular Vesicles by TEM Using

Frederic St-Denis-Bissonnette1,2, Barry Ngo1,2, Hala Halabi1,2

  • 1Biologic and Radiopharmaceutical Drugs Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, ON K1A 0K9, Canada.

Langmuir : the ACS Journal of Surfaces and Colloids
|December 27, 2025
PubMed
Summary

Researchers developed an optimized positive-staining protocol for transmission electron microscopy (TEM) of extracellular vesicles (EVs). This method enhances image contrast and preserves EV morphology, offering a reliable framework for structural analysis.

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Area of Science:

  • Nanotechnology
  • Biophysics
  • Microscopy

Background:

  • Transmission electron microscopy (TEM) is crucial for visualizing extracellular vesicles (EVs) due to its high resolution.
  • Current TEM sample preparation protocols for EVs often lack detailed methodology, leading to inconsistent results.

Purpose of the Study:

  • To report an optimized positive-staining protocol for TEM-based visualization of extracellular vesicles (EVs).
  • To enhance image contrast, preserve EV morphology, and reduce background artifacts in TEM imaging.

Main Methods:

  • Sequential application of Uranyless and lead citrate stains with an intermediate buffered wash.
  • Confirmation of staining specificity and elemental localization using Energy-Dispersive X-ray Spectroscopy (EDS).

Main Results:

  • The optimized protocol significantly improves image contrast and preserves the morphology of extracellular vesicles.
  • Reduced background artifacts were observed, leading to clearer visualization of EVs.
  • The protocol demonstrated reproducibility and robustness in structural analysis.

Conclusions:

  • The developed positive-staining method provides a reliable framework for TEM-based extracellular vesicle imaging.
  • This protocol can serve as a reference standard for high-quality TEM analysis of EVs.