Resolution and Limitations in Biological Applications of Atomic Force Microscopy

  • Jie Yang
  • Lukas K. Tamm
  • Zhifeng Shao


Atomic force microscopy (AFM) has been applied to image DNA and a membrane protein: cholera toxin. By use of the Kleinschmidt method, DNA molecules were picked up on carbon-coated mica surfaces and imaged by AFM in air and in organic solvents. The resolution was found to be closely related to the adhesion force and a resolution of 3–6 nm was routinely obtained when the adhesion force was below 3 nN. The role of the adhesion force, the tip condition and the specimen preparation on resolution and imaging quality will be discussed. Polymerized diacetylene phosphatidylcholine (DAPC) bilayers provide a relatively stable matrix for studying membrane proteins. When cholera toxin (complete or B-subunit oligomer) was bound to mixed bilayers of DAPC and the receptor glycolipid GM1, the subunit structure was well resolved by AFM in buffer, without crystallization. The resolution was better than 2 nm with excellent reproducibility for a probe force of 0.3–0.5 nN. These results show that individual biomacromolecules under native conditions can be imaged by AFM with high spatial resolution.


Adhesion Force Cholera Toxin High Resolution Image Lateral Resolution Probe Force 
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  1. 1.
    T.R. Albrecht, S. Akamine, T.E. Carver, and C.F. Quate, Microfabrication of cantilever styli for the atomic force microscope, J. Vac. Sci. Technol. A8, 3386–3396 (1990).CrossRefGoogle Scholar
  2. 2.
    G. Binnig, C.F. Quate, and C.H. Gerber, Atomic Force Microscope, Phys. Rev. Lett. 56, 930–933 (1986).CrossRefGoogle Scholar
  3. 3.
    C. Bustamante, J. Vesenka, C.L. Tang, W. Rees, M. Guthod, and R. Keller, Circular DNA molecules imaged in air by scanning force microscopy, Biochemistry 31, 22–26 (1992).CrossRefGoogle Scholar
  4. 4.
    H.-J. Butt, K.H. Downing, and P.K. Hansma, Imaging the membrane protein bacteriorhodopsin with the atomic force microscope, Biophys. J. 58, 1473–1480 (1990).CrossRefGoogle Scholar
  5. 5.
    H.-J. Butt, E.K. Wolff, S.A.C. Gould, B.D. Northern, C.M. Peterson, and P.K. Hansma, Imaging cells with the atomic force microscope, J. Struct. Biol. 105, 54–61 (1990).Google Scholar
  6. 6.
    K.L. Dorrington, The theory of viscoelasticity in biomaterials, in: “The Mechanical Properties of Biological Materials,” Cambridge University Press, Cambridge (1979).Google Scholar
  7. 7.
    D.M. Gill, Mechanism of action of cholera toxin, Adv. Cyc. Nucleo. Res. 8, 85–118 (1977).Google Scholar
  8. 8.
    W. Haberle, J.K.H. Horber, and G. Binnig, Force microscopy of living cells, J. Vac. Sci. Techno. 9, 1210–1213 (1991).CrossRefGoogle Scholar
  9. 9.
    W. IIaberle, J.K.H. Horber, F. Ohnesorge, D.P.E. Smith, G. Binnig, In situ investigation of single living cells infected by viruses, Ultramicroscopy 42–44, 1161–1167 (1992).Google Scholar
  10. 10.
    H.G. Hansma, R.L. Sinsheimer, M.-Q. Li, and P.K. Hansma, Atomic force microscopy of single-and double-stranded DNA, Nucleic. Acids Res. 20, 3585–3590 (1992).CrossRefGoogle Scholar
  11. 11.
    H.G. Hansma, J. Vesenka, C. Siegerist, G. Kelderman, H. Morrett, P.L. Sinsheimer, V. Flings, C. Bustamante, P.K. Hansma, Reproducible imaging and dissection of plasmid DNA under liquid with atomic force microscopy, Science 256, 1180–1184 (1992).CrossRefGoogle Scholar
  12. 12.
    E. Henderson, Imaging and nanodissection of individual supercoiled plasmid by atomic force microscopy, Nucleic Acids Res. 20, 445–447 (1992).CrossRefGoogle Scholar
  13. 13.
    E. Ilenderson, P.G. Hayelon, and D.S. Sakaguchi, Actin filament dynamics in living glial cells imaged by atomic force microscopy, Science 257, 1944–1946 (1992).CrossRefGoogle Scholar
  14. 14.
    J.H. Hoh, R. I,al, S.A. John, J.-P. Revel, and M.F. Arnsdorf, Atomic force microscopy and dissection of gap junctions, Science 253, 1405–1408 (1991).CrossRefGoogle Scholar
  15. 15.
    J. Holmgren, Actions of cholera toxin and the prevention and treatment of cholera, Nature 292, 413417 (1981).Google Scholar
  16. 16.
    D. Johnston, S. Sanghera, M. Pons, and D. Chapman, Phospholipid polymers-sythesis and spectral characteristics, Biochim. Biophys. Acta 602, 57–69 (1980).CrossRefGoogle Scholar
  17. 17.
    Y.L. Lyubchenko, A.A. Gall, L.S. Shlyakhtenko, R.E. Harrington, and S.M. Lindsay, Atomic force microscopy imaging of large double stranded DNA molecules, Biophys. J. 61, A149 (1992).Google Scholar
  18. 18.
    Y.L. Lyubchenko, B. L. Jacobs, and S.M. Lindsay, Atomic force microscopy of reovirus dsRNA: a routine technique for length measurements, Nucleic Acids Res. 20, 3983–3986 (1992).CrossRefGoogle Scholar
  19. 19.
    Y.I,. Lyubchenko, P.I. Oden, D. Lampner, S.M. Lindsay and K.A. Dunker, Atomic force microscopy of DNA and bacteriophage in air, water and propanol: the role of adhesion forces, Nucl. Acids Res. in press.Google Scholar
  20. 20.
    G. Mosser, and A. Brisson, Structural analysis of two-dimensional arrays of cholera toxin B-subunit, J. Electron Micro. Tech. 18, 387–394 (1991).CrossRefGoogle Scholar
  21. 21.
    J. Mou, J. Yang, and Z. Shao, An optical detection low temperature atomic force microscope at ambient pressure for biological research, Rev. Sci. Instrum. in press.Google Scholar
  22. 22.
    M. Radmacher, R.W. Tillmann, M. Fritz, and H.F. Gaub, From molecules to cells: imaging soft samples with the atomic force microscope, Science 257, 1900–1905 (1992).CrossRefGoogle Scholar
  23. 23.
    R.A. Reed, J. Mattai, and G.G. Shipley, Interaction of cholera toxin with ganglioside GM, receptors in supported lipid monolayers, Biochemistry 26, 824–832 (1987).CrossRefGoogle Scholar
  24. 24.
    D.G. Rhodes, A. Xu, and R. Bittman, Structure of polymerizable lipid bilayers V: synthesis, bilayer structure and proterties of diacetylenic ether and ester lipids, Biochim. Biophys. Acta 1128, 93–104 (1992).CrossRefGoogle Scholar
  25. H.O. Ribi, D.S. Ludwig, K.L. Mercer, G.K. Schoolnik, R.D. Kornberg, Three-dimensional structure of cholera toxin penetrating a lipid membrane, Science 239, 1272–1276 (1988).Google Scholar
  26. 26.
    D. Rugar, and P. Hansma, Atomic force microscopy, Physics Today 43, 23–30 (1990).CrossRefGoogle Scholar
  27. 27.
    D. Sarid, “Scanning Force Microscopy,” Oxford University Press, Oxford, New York (1990).Google Scholar
  28. 28.
    T. Tomie, H. Shimizu, T. Majima, M. Yamada, T. Kanayama, H. Kondo, M. Yano, and M. Ono, Three-dimensional readout of flash X-ray images of living sperm in water by atomic force microscopy, Science 252, 691–693 (1991).CrossRefGoogle Scholar
  29. 29.
    J. Vesenka, M. Guthold, C.L. Tang, D. Keller, E. Delaine, and C. Bustamante, Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope, Ultrmicroscopy 42–44, 1243–1249 (1992).CrossRefGoogle Scholar
  30. 30.
    H. Ximen, and P.E. Russell, Microfabrication of AFM tips using focused ion and electron beam techniques, Ultramicroscopy 42–44, 1526–1532 (1992).CrossRefGoogle Scholar
  31. 31.
    J. Yang and Z. Shao, The effect of probe force on the resolution of atomic force microscopy of DNA. Ultramicroscopy, in press.Google Scholar
  32. 32.
    J. Yang, A.V. Somlyo, M.K. Reedy, K. Takeyasu, L.K. Tamm, M. Allietta, T.W. Tillack, and Z. Shao, Biological applications of AFM, in: “Proc. 50th EMSA Annual Meeting,” Boston, MA., 1138–1139 (1992).Google Scholar
  33. 33.
    J. Yang, K. Takeyasu and Z. Shao, Atomic force microscopy of DNA molecules, FEES Lett. 301, 173–176 (1992).CrossRefGoogle Scholar
  34. 34.
    J. Yang, L.K. Tamm, T.W. Tillack, Z. Shao, New Approach for Atomic Force Microscopy of Membrane Proteins: the Imaging of Cholera Toxin, J. Mol. Biol. 229, 286–290 (1993).CrossRefGoogle Scholar
  35. 35.
    J.A.N. 7,asadzinski, C.A. IIelm, M.I,. Longo, A.L. Weisenhorn, S.A.C. Gould, and P.K. Hansma, Atomic force microscopy of hydrated phosphatidylethanolamine, Biophys. J. 59, 755–760 (1991).CrossRefGoogle Scholar
  36. 36.
    F. Zenhausern, M. Adriah, I3.T. Heggeler-Bordier, R. Emch, M. Jobin, M. Taborclli, P. Descouts, Imaging of DNA by scanning force microscopy, J. Struct. Biol. 108, 69–73 (1992).CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1994

Authors and Affiliations

  • Jie Yang
    • 1
  • Lukas K. Tamm
    • 1
  • Zhifeng Shao
    • 1
  1. 1.Department of Molecular Physiology and Biological PhysicsUniversity of VirginiaCharlottesvilleUSA

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