A recent study published in the Journal of Extracellular Vesicles explored how high-speed atomic force microscopy (HS-AFM) videography can be used to analyze small extracellular vesicles (sEVs) at the nanoscale.
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These vesicles play important roles in cell-to-cell communication and are promising targets for disease diagnosis, particularly in cancer.
The researchers focused on understanding how sEVs interact with surface markers to better define their biological functions and potential as biomarkers.
Advances in Imaging Technology
Extracellular vesicles (EVs) are tiny, membrane-enclosed particles released by cells. They are involved in many biological processes, including cell communication, disease progression, and drug delivery. Types of EVs include exosomes, microvesicles, and apoptotic bodies, which carry proteins, lipids, and nucleic acids.
EVs have received attention as potential biomarkers, especially for cancer diagnosis. However, traditional imaging methods like confocal and electron microscopy, nanoparticle tracking analysis (NTA), and flow cytometry have limitations. These include low resolution, sample preparation artifacts, and changes in vesicle shape. Such challenges highlight the need for tools that can capture the structure of EVs more accurately.
HS-AFM offers an advantage by enabling real-time imaging of biological samples in near-native conditions. It uses an oscillating cantilever tip to scan surfaces and detect nanoscale features without the need for extensive processing. This makes it well suited for studying sEVs, which range from 40 to 160 nanometers in size. HS-AFM allows researchers to observe their structure and interactions with high precision.
Implementing HS-AFM Videography Technology
In this study, the researchers used HS-AFM to distinguish between two types of sEVs: those enriched with tetraspanins (exosome-like) and those lacking them (ectosome-like). The vesicles were derived from HEK293T cells.
First, the researchers cultured cells and collected the media after four days. They removed cells and debris by centrifugation and filtration through a 0.22-µm membrane membrane. sEVs were isolated using ultracentrifugation and a Tim-4 affinity-based method that binds to phosphatidylserine on the vesicle membrane.
To examine surface markers, the researchers labeled the sEVs with antibodies against CD63 and CD81. NTA was used to measure the size and concentration of the vesicles. HS-AFM imaging was then performed under near-physiological conditions using a custom-built setup equipped with a cantilever and laser-based detection. A gentle tapping force helped preserve the shape of the vesicles during imaging.
Advanced image analysis tools, such as ImageJ and Gwyddion, were used to reconstruct 3D shapes and measure spatial features. This approach allowed the team to study sEV structure and behavior in real time.
Key Findings: What HS-AFM Revealed
The outcomes showed significant differences in the structural dynamics of sEVs, identifying two distinct subpopulations: those with diameters (d) ≤100 nm and those >100 nm. Specifically, sEVs with d > 100 nm exhibited an average height of 43.98 ± 12.77 nm, a diameter of 120 ± 16 nm, and a volume of 480,029 ± 249,055 nm3. In contrast, sEVs with d ≤ 100 nm had an average height of 28.79 ± 8.02 nm, a diameter of 67 ± 14 nm, and a volume of 108,799 ± 72,670 nm3. The smaller vesicles displayed reduced height fluctuations, indicating greater membrane rigidity than their larger counterparts.
The study also demonstrated that antibodies targeting tetraspanins CD63 and CD81 predominantly co-localized with sEVs ≤ 100 nm, suggesting selective enrichment of these markers. This pattern supports that these sEVs primarily originate from endosomal pathways, while larger ones may arise from the plasma membrane. These distinctions are crucial for understanding the intracellular origins and diagnostic relevance of sEV subtypes.
Additionally, HS-AFM videography enabled real-time visualization of antibody-sEV interactions. Immunoglobulin G (IgG) molecules against CD63 and CD81 exhibited conformational flexibility based on proximity to target antigens, enhancing binding efficiency. In contrast, the control antibody exhibited minimal interaction. The stable attachment of IgG CD63 and IgG CD81 to the vesicle surface highlighted the potential of HS-AFM to explore the functional and dynamic properties of sEVs under near-physiological conditions.
Potential Applications in Medicine and Research
This work could strongly impact diagnostics, therapeutics, and biomarker development. Being able to profile sEVs more precisely could help identify subtypes linked to specific diseases, including cancer. This information could improve early detection and monitoring using non-invasive techniques.
Because HS-AFM preserves the natural structure of vesicles and doesn’t require labels, it is well-suited for clinical studies. It also helps researchers better understand how sEVs are formed and how they function in cell communication. This knowledge could support the development of targeted therapies.
Combining HS-AFM with antibody-based profiling (immunophenotyping) may improve the accuracy of vesicle characterization in patient samples. This would support efforts in personalized medicine by helping tailor treatments based on individual biological markers.
Conclusion and Future Directions
The study showed that HS-AFM videography is a useful tool for analyzing sEVs in real time. It provided new insights into the structure, behavior, and surface markers of different vesicle subtypes.
As the technology develops, it may help improve how we detect, classify, and study sEVs. This could support new approaches in disease diagnosis and targeted treatments. Future work should aim to increase the speed of data collection, combine HS-AFM with other methods, and explore how to apply this approach in clinical settings.
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Journal Reference
Sandira, M I., et al. (2025). Nanoscopic Profiling of Small Extracellular Vesicles via High-Speed Atomic Force Microscopy (HS-AFM) Videography. Journal of Extracellular Vesicles, e270050. DOI: 10.1002/jev2.70050, https://isevjournals.onlinelibrary.wiley.com/doi/10.1002/jev2.70050
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