Why use AFM to image DNA?

Imaging DNA with an atomic force microscope is a common application. The sample preparation is very straightforward and with the HR-AFM the imaging can be done at far lower cost than with other microscopes. AFMs can directly image DNA on a flat surface such as mica, typically bound to it through a divalent cation such as Mg2+ or Ni2+, washed and air-dried. You can also do these measurements in liquid, but scanning in air is far easier and a good place to start.

The purity and concentration of the DNA starting material is critically important, together with clean reagents and handling during sample preparation. And of course since we’re imaging a nanoscale molecule, a low noise floor is key.

AFM image of mixed genomic DNA purified from a soil sample.

Topography image of genomic DNA, adsorbed on mica with 10 mM NiCl2, dried and imaged in vibrating mode in air.

Examples on AFMWorkshop microscopes

Once a high-quality sample is prepared, generating great DNA images is a question of reducing noise from the environment and by correctly adjusting the imaging parameters.

An interesting example that illustrates the different structures DNA can form in the bacteriophage M13, a popular tool in molecular biology. M13 DNA is is commercially available in both single-strand and double-stranded form. Its imaging by AFM illustrates the dramatic differences in the secondary structure of ssDNA and dsDNA.

AFM image of M13 bacteriophage single-stranded DNA, 4x4 um scan, topography image with color indexed height
AFM image of M13 bacteriophage single-stranded DNA, 2x2 um scan, topography image with color indexed height

Topography images of single-stranded M13mp18 DNA illustrates how ssDNA folds in tight secondary structures (left: 4×4 um scan, right: 2×2 um). The DNA was adsorbed on mica with 10 mM MgCl2, dried, and imaged on an AFMWorkshop HR-AFM with <35 pm noise floor.

AFM image of M13 bacteriophage double-stranded DNA, 5x5 um scan, topography image with color indexed height
AFM image of M13 bacteriophage double-stranded DNA, 2x2 um scan, topography image with color indexed height

The double-stranded M13mp18 DNA has the typical appearance of dsDNA plasmids with varying degrees of supercoiling (left: 5×5 um scan, right: 2×2 um). The DNA was adsorbed on mica with 10 mM MgCl2, dried, and imaged on an AFMWorkshop HR-AFM with <35 pm noise floor.

AFM image of M13 bacteriophage single-stranded DNA, 2x2 um scan, 3d rendered and shaded
AFM image of M13 bacteriophage double-stranded DNA, 2x2 um scan, 3d rendered and shaded

The same M13 ssDNA and dsDNA images 3d rendered and shaded.

More high-resolution topography images of DNA from an AFMWorkshop HR-AFM.

DNA on mica terraces imaged with an HR-AFM
circular DNA aggregates imaged with an HR-AFM
1x1 um scan circular DNA imaged with an HR-AFM showing fragmentation of the DNA
circular DNA imaged with an HR-AFM showing fragmentation of the DNA

More high-resolution topography images of DNA from an AFMWorkshop TT-2-AFM (69 pm noise floor).

AFM image of mixed genomic DNA purified from a soil sample.
genomic DNA applied on mica at too high concentration

Mixed genomic DNA purified from a soil sample. 

DNA applied to mica at too high concentration.

fork in a DNA fragment with protein caps and line profiles for height measurements

Topography image cropped from the image above, showing an apparent fork in a linear DNA fragment decorated with protein caps. The line profiles show the measured height of the double-stranded DNA (red) and the protein caps (black). Images were taken on an AFMWorkshop TT-2 AFM with 69 pm noise floor.

Stay tuned, we’re generating more interesting DNA data!

Literature and protocols

  • Hansma, H. G.; Revenko, I.; Kim, K.; Laney, D. E. Atomic Force Microscopy of Long and Short Double-Stranded, Single-Stranded and Triple-Stranded Nucleic Acids. Nucleic Acids Res. 1996, 24 (4), 713–720. https://doi.org/10.1093/nar/24.4.713.

  • Main, K. H. S.; Provan, J. I.; Haynes, P. J.; Wells, G.; Hartley, J. A.; Pyne, A. L. B. Atomic Force Microscopy—A Tool for Structural and Translational DNA Research. APL Bioeng. 2021, 5 (3), 031504. https://doi.org/10.1063/5.0054294.

  • Lyubchenko, Y. L. Preparation of DNA and Nucleoprotein Samples for AFM Imaging. Micron Oxf. Engl. 1993 2011, 42 (2), 196–206. https://doi.org/10.1016/j.micron.2010.08.011.

  • Lyubchenko, Y. L.; Shlyakhtenko, L. S. Imaging of DNA and Protein–DNA Complexes with Atomic Force Microscopy. Crit. Rev. Eukaryot. Gene Expr. 2016, 26 (1), 63–96. https://doi.org/10.1615/CritRevEukaryotGeneExpr.v26.i1.70.

  • Fotiadis, D.; Scheuring, S.; Müller, S. A.; Engel, A.; Müller, D. J. Imaging and Manipulation of Biological Structures with the AFM. Micron 2002, 33 (4), 385–397. https://doi.org/10.1016/S0968-4328(01)00026-9.

  • Hamon, L., Pastré, D., Dupaigne, P., Le Breton, C., Le Cam, E., & Piétrement, O. (2007). High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein–DNA complexes. Nucleic acids research35(8), e58. https://doi.org/10.1093/nar/gkm147

  • Fuentes-Perez, M. E., Dillingham, M. S., & Moreno-Herrero, F. (2013). AFM volumetric methods for the characterization of proteins and nucleic acids. Methods (San Diego, Calif.)60(2), 113–121. https://doi.org/10.1016/j.ymeth.2013.02.005