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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
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.
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.
