Imaging viruses with atomic force microscopy

Why use AFM to image viruses?

In this application, AFM can complement information from transmission electron microscopy (TEM), dynamic light scattering (DLS), and other methods that are used to characterize virus particle morphology and statistical properties. AFM falls between these methods, providing quantitative 3D data from which statistical quantities can be extracted.

Straightforward sample preparation: Liquid to dry sample to image in ~30 minutes
Small sample volume: 1-5 µL
No hazardous reagents
Low cost: <$30 in consumables, <$65k AFM (MolBio3D or HR-AFM)
Automated extraction of statistical quantities
Advanced experiments can be performed in buffer (including mechanobiology measurements).

Example: Adeno-associated virus (AAV)

Adeno-associated virus (AAV) is a prominent example due to its widespread use in gene therapy. AAV capsids are approximately 26 nm in diameter and contain a linear single-stranded DNA (ssDNA) genome of approximately 4.8 kb.

AFM image of Adeno-associated virus (AAV) capsids

AAV particles deposited on poly-L-lysine and dried, illustrating particle height distribution.

AFM image of Adeno-associated virus (AAV) capsids, color indexed to highlight released viral ssDNA

The same image of AAV particles height indexed to highlight released viral ssDNA (red aggregates). Note that some ssDNA is closely associated with capsids.

AAV capsids can be segmented automatically to extract statistical quantities.

Automated segmentation of AAV particles in a 4×4 um scan to extract statistical quantities. Our software performs segmentation, data extraction and visualizations directly from raw data.

Maximum height distribution of AAV particles, extracted automatically from AFM data

Extracted AAV particle height distribution.

Extracted AAV particle volume distribution.

AAV particles major axis length distribution

Extracted AAV particle major axis length distribution (particle shape).

AAV particles minor axis length distribution

Extracted AAV particle minor axis length distribution (particle shape).

Note that particle height is influenced by how strongly the capsids collapse during the adhesion and drying process, whereas the lateral dimensions are influenced by AFM tip convolution.

Literature

Horowitz, E. D., Rahman, K. S., Bower, B. D., Dismuke, D. J., Falvo, M. R., Griffith, J. D., Harvey, S. C., & Asokan, A. (2013). Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release. Journal of virology, 87(6), 2994–3002. https://doi.org/10.1128/JVI.03017-12
Zeng, C., Moller-Tank, S., Asokan, A., & Dragnea, B. (2017). Probing the Link among Genomic Cargo, Contact Mechanics, and Nanoindentation in Recombinant Adeno-Associated Virus 2. The journal of physical chemistry. B, 121(8), 1843–1853. https://doi.org/10.1021/acs.jpcb.6b10131
Heldt, C. L., Areo, O., Joshi, P. U., Mi, X., Ivanova, Y., & Berrill, A. (2023). Empty and Full AAV Capsid Charge and Hydrophobicity Differences Measured with Single-Particle AFM. Langmuir : the ACS journal of surfaces and colloids, 39(16), 5641–5648. https://doi.org/10.1021/acs.langmuir.2c02643
Zoratto, S., Weiss, V. U., Friedbacher, G., Buengener, C., Pletzenauer, R., Foettinger-Vacha, A., Graninger, M., & Allmaier, G. (2021). Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy. ACS omega, 6(25), 16428–16437. https://doi.org/10.1021/acsomega.1c01443
Rossi, A., Dupaty, L., Aillot, L., Zhang, L., Gallien, C., Hallek, M., Odenthal, M., Adriouch, S., Salvetti, A., & Büning, H. (2019). Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity. Scientific reports, 9(1), 3631. https://doi.org/10.1038/s41598-019-40071-1
Bernaud, J., Rossi, A., Fis, A., Gardette, L., Aillot, L., Büning, H., Castelnovo, M., Salvetti, A., & Faivre-Moskalenko, C. (2018). Characterization of AAV vector particle stability at the single-capsid level. Journal of biological physics, 44(2), 181–194. https://doi.org/10.1007/s10867-018-9488-5