Nanoimaging pp 315-341

Part of the Methods in Molecular Biology book series (MIMB, volume 950) | Cite as

Atomic Force Microscopy Imaging of Macromolecular Complexes



This chapter reviews amplitude modulation (AM) AFM in air and its applications to high-resolution imaging and interpretation of macromolecular complexes. We discuss single DNA molecular imaging and DNA–protein interactions, such as those with topoisomerases and RNA polymerase. We show how relative humidity can have a major influence on resolution and contrast and how it can also affect conformational switching of supercoiled DNA. Four regimes of AFM tip–sample interaction in air are defined and described, and relate to water perturbation and/or intermittent mechanical contact of the tip with either the molecular sample or the surface. Precise control and understanding of the AFM operational parameters is shown to allow the user to switch between these different regimes: an interpretation of the origins of topographical contrast is given for each regime. Perpetual water contact is shown to lead to a high-resolution mode of operation, which we term SASS (small amplitude small set-point) imaging, and which maximizes resolution while greatly decreasing tip and sample wear and any noise due to perturbation of the surface water. Thus, this chapter provides sufficient information to reliably control the AFM in the AM AFM mode of operation in order to image both heterogeneous samples and single macromolecules including complexes, with high resolution and with reproducibility. A brief introduction to AFM, its versatility and applications to biology is also given while providing references to key work and general reviews in the field.

Key words

AFM Biomolecules Macromolecular complexes Double-stranded DNA RNA polymerase Topoisomerase Water Height 


  1. 1.
    Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phy Rev Lett 56:930–933CrossRefGoogle Scholar
  2. 2.
    Quate CF (1994) The AFM as a tool for surface imaging. Surf Sci 299–300:980–995CrossRefGoogle Scholar
  3. 3.
    Bustamante C, Keller D (1995) Scanning force microscopy in biology. Physics today 48:33–38CrossRefGoogle Scholar
  4. 4.
    Hansma HG, Hoh JH (1994) Biomolecular imaging with the atomic force microscope. Ann Rev Biophy Biomol Struc 23:115–140CrossRefGoogle Scholar
  5. 5.
    Garcia R, Perez R (2002) Dynamic atomic force microscopy methods. Surf Sci Rep 47:197–301CrossRefGoogle Scholar
  6. 6.
    Giessibl FJ (2003) Advances in atomic force microscopy. Rev Modern Physics 75:949–983CrossRefGoogle Scholar
  7. 7.
    Israelachvili J (1991) Intermol. & Surf. Forces. Academic, New YorkGoogle Scholar
  8. 8.
    Kienbergera F, Costab TL, Zhua R, Kadad G, Reithmayere M, Chtcheglovaa L, Rankla C, Pachecof BFA, Thalhammerb S et al (2007) Dynamic force microscopy imaging of plasmid DNA and viral RNA. Biomaterials 28:2403–2411CrossRefGoogle Scholar
  9. 9.
    Zhong Q, Innlss D, Kjoller K, Elings VB (1993) Fractured polymer/silica fiber surface studied by tapping mode atomic force microscopy. Surf Sci Lett 290:L688–L692CrossRefGoogle Scholar
  10. 10.
    Hansma PK, Cleveland JP, Radmacher M et al (1994) Tapping mode atomic force microscopy in liquids. Appl Phy Lett 64:1738–1740CrossRefGoogle Scholar
  11. 11.
    Sahin O, Magonov S, Su C, Quate CF, Solgaard O (2007) An atomic force microscope tip designed to measure time-varying nanomechanical forces. Nat Nanotech 2:507–514CrossRefGoogle Scholar
  12. 12.
    Proksch R (2006) Multi-frequency, repulsive mode amplitude modulated atomic force microscopy. Appl Phys Lett 89:113121–113123CrossRefGoogle Scholar
  13. 13.
    Francis LW, Lewis PD, Wright CJ, Conlan RS (2010) Atomic force microscopy comes of age. Biol Cell 102:133–143Google Scholar
  14. 14.
    Parot P, Dufrene YF, Hinterdorfer P et al (2007) Past, present and future of atomic force microscopy in life sciences and medicine. J Mol Recog 20:418–431CrossRefGoogle Scholar
  15. 15.
    Giessibl FJ (1995) Atomic Resolution of the Silicon (111)-(7x7) Surface by Atomic Force Microscopy. Science 267:68–71PubMedCrossRefGoogle Scholar
  16. 16.
    Weisenhorn AL, Hansma PK, Albrecht TR, Quate CF (1989) Forces in atomic force microscopy in air and water. Appl Phys Lett 54:2651–2653CrossRefGoogle Scholar
  17. 17.
    Tamayo J, Garcia R (1996) Deformation, contact time, and phase contrast in tapping mode scanning force microscopy. Langmuir 12:4430–4435CrossRefGoogle Scholar
  18. 18.
    Kiracofe D, Raman A (2010) On eigenmodes, stiffness, and sensitivity of atomic force microscope cantilevers in air versus liquids. J Appl Phys 107:033506–033515CrossRefGoogle Scholar
  19. 19.
    Turner RD, Kirkham J, Devine D, Thomson NH (2009) Second harmonic atomic force microscopy of living Staphylococcus aureus bacteria Appl. Phys Lett 94:043901Google Scholar
  20. 20.
    Stark RW (2004) Spectroscopy of higher harmonics in dynamic atomic force microscopy. Nanotechnology 15:347–351CrossRefGoogle Scholar
  21. 21.
    Patil S, Martinez NF, Lozano JR, Garcia R (2007) Force microscopy imaging of individual protein molecules with sub-pico Newton force sensitivity. J Mol Recogn 20:516–523CrossRefGoogle Scholar
  22. 22.
    Xu X, Melcher J, Basak S, Reifenberger R, Raman A (2009) Compositional contrast of biological materials in liquids using the momentary excitation of higher eigenmodes in dynamic atomic force microscopy. Phys Rev Lett 102:060801–060804PubMedCrossRefGoogle Scholar
  23. 23.
    Melcher J, Carrasco C, Xu X et al (2009) Origins of phase contrast in the atomic force microscope in liquids. Proc Natl Acad Sci USA 106:13655–13660PubMedCrossRefGoogle Scholar
  24. 24.
    Basak S, Raman A, Garimella SV (2006) Hydrodynamic loading of microcantilevers vibrating in viscous fluids. J Appl Phys 99:114906–114915CrossRefGoogle Scholar
  25. 25.
    Alessandrini A, Facci P (2005) AFM: a versatile tool in biophysics. Meas Sci Techn 16:R65–R92CrossRefGoogle Scholar
  26. 26.
    Gould S, Marti O, Drake B et al (1988) Molecular resolution images of amino acid crystals with the atomic force microscope. Nature 332:332–334CrossRefGoogle Scholar
  27. 27.
    Albrecht TR, Grutter P, Horne D, Rugar D (1991) Frequency modulation detection using high‐Q cantilevers for enhanced force microscope sensitivity. J Appl Phys 69:668–673CrossRefGoogle Scholar
  28. 28.
    Martin Y, Williams CC, Wickramasinghe HK (1987) Atomic force microscope-force mapping and profiling on a sub 100-Ǻ scale. J Appl Phys 61:4723–4729CrossRefGoogle Scholar
  29. 29.
    Santos S (2011) PhD thesis: Dynamic atomic force microscopy and its applications in biomolecular imaging. University of LeedsGoogle Scholar
  30. 30.
    Drake B, Prater CB, Weisenhorn AL et al (1989) Imaging crystals, polymers, and processes in water with the atomic force microscope. Science 243:1586PubMedCrossRefGoogle Scholar
  31. 31.
    Kodera N, Yamamoto D, Ishikawa R, Ando T (2010) Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468:72–76PubMedCrossRefGoogle Scholar
  32. 32.
    Gross L, Mohn F, Moll N, Liljeroth P, Meyer G (2009) The Chemical Structure of a Molecule Resolved by Atomic Force Microscopy. Science 325:1110–1114PubMedCrossRefGoogle Scholar
  33. 33.
    Gan Y (2009) Atomic and subnanometer resolution in ambient conditions by atomic force microscopy. Surf Sci Rep 64:99–121CrossRefGoogle Scholar
  34. 34.
    Santos S, Thomson NH (2011) High resolution imaging of Immunoglobulin G (IgG) antibodies and other biomolecules using amplitude modulation atomic force microscopy in air. Humana Press, New YorkGoogle Scholar
  35. 35.
    Gerber C, Lang HP (2006) How the doors to the nanoworld were opened. Nat Nanotechn 1:3–5CrossRefGoogle Scholar
  36. 36.
    Garcia R, Magerele R, Perez R (2007) Nanoscale compositional mapping with gentle forces. Nat Mater 6:405–411PubMedCrossRefGoogle Scholar
  37. 37.
    Ostendorf F, Schmitz C, Hirth S et al (2008) How flat is an air-cleaved mica surface? Nanotechnology 19:305705PubMedCrossRefGoogle Scholar
  38. 38.
    Bezanilla M, Manne S, Laney DE, Lyubchenko YL, Hansma HG (1995) Adsorption of DNA to mica, silylated mica, and minerals: characterization by atomic force microscopy. Langmuir 11:655–659CrossRefGoogle Scholar
  39. 39.
    Hansma H, Laney D (1996) DNA binding to mica correlates with cationic radius: assay by atomic force microscopy. Biophysical J 70:1933–1939CrossRefGoogle Scholar
  40. 40.
    Vesenka J, Guthold M, Tang CL, Keller D, Delaine E, Bustamante C (1992) Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope. Ultramicroscopy 42–44:1243–1249PubMedCrossRefGoogle Scholar
  41. 41.
    Ostendorf F, Schmitz C, Hirth S et al (2009) Evidence for potassium carbonate crystallites on Air-cleaved mica surfaces. Langmuir 25:10764–10767PubMedCrossRefGoogle Scholar
  42. 42.
    Santos S, Verdaguer A, Souier T, Thomson HN, Chiesa M (2011) Measuring the true height of water layers in the nanoscale. Nanotechnology 22:465705–465713PubMedCrossRefGoogle Scholar
  43. 43.
    Billingsley DJ, Kirkham J, Bonass WA, Thomson NH (2010) Atomic force microscopy of DNA at high humidity: irreversible conformational switching of supercoiled molecules. Phys Chem Chem Phys 12:14727–14734PubMedCrossRefGoogle Scholar
  44. 44.
    Binnig G, Rohrer H. 1984. Scanning tunneling microscopy. In Trends in Physics, Ed. J Janta, J Pantoflicek, pp. 38-46. The Hague: Eur. Phys. SocGoogle Scholar
  45. 45.
    Garcia R, San Paulo A (1999) Attractive and repulsive tip-sample interaction regimes in tapping mode atomic force microscopy. Phys Rev B 60:4961–4967CrossRefGoogle Scholar
  46. 46.
    Santos S, Barcons V, Font J, Thomson NH (2010) Bi-stability of amplitude modulation AFM in air: deterministic and stochastic outcomes for imaging biomolecular systems. Nanotechnology 21:225710–225720PubMedCrossRefGoogle Scholar
  47. 47.
    Santos S, Barcons V, Font J, Thomson NH (2010) Cantilever dynamics in amplitude modulation AFM: continuous and discontinuous transitions. J Phys D: Appl Phys 43:275401–275407CrossRefGoogle Scholar
  48. 48.
    Stark RW (2010) Bistability, higher harmonics, and chaos in AFM Materials Today (2010) 13(9):24–32 Google Scholar
  49. 49.
    Tamayo J, Garcia R (1998) Relationship between phase shift and energy dissipation in tapping-mode scanning force microscopy. Appl Phys Lett 73:2926–2928CrossRefGoogle Scholar
  50. 50.
    Santos S, Barcons V, Verdaguer A et al (2011) How localised are energy dissipation processes in the nanoscale? Nanotechnology 22:345401–345407PubMedCrossRefGoogle Scholar
  51. 51.
    Zitzler L, Herminghaus S, Mugele F (2002) Capillary forces in tapping mode atomic force microscopy. Phys Rev B 66:155436–155438CrossRefGoogle Scholar
  52. 52.
    Garcia R, San Paulo A (2000) Dynamics of a vibrating tip near or in intermittent contact with a surface. Phys Rev B 61:R13381–R13384CrossRefGoogle Scholar
  53. 53.
    Marth M, Maier D, Honerkamp J (1999) A unifying view on some experimental effects in tapping-mode atomic force microscopy. J Appl Phys 85:7030–7036CrossRefGoogle Scholar
  54. 54.
    Santos S, Barcons V, Verdaguer A, Chiesa M (2011) Sub-harmonic excitation in AM AFM in the presence of adsorbed water layers. J Appl Phys 110:114902–114911CrossRefGoogle Scholar
  55. 55.
    Couturier G, Boisgard R, Nony L, Aimé JP (2003) Noncontact atomic force microscopy: Stability criterion and dynamical responses of the shift of frequency and damping signal. Rev Scientific Instrum 74:2726–2734CrossRefGoogle Scholar
  56. 56.
    Garcia R, San PA (2000) Amplitude curves and operating regimes in dynamic atomic force microscopy. Ultramicroscopy 82:79–83PubMedCrossRefGoogle Scholar
  57. 57.
    Santos S, Barcons V, Christenson HK, Font J, Thomson NH (2011) The intrinsic resolution limit in the atomic force microscope: implications for heights of nano-scale features. PLoS One 6:e23821PubMedCrossRefGoogle Scholar
  58. 58.
    Moreno-Herrero F, Colchero J, Baro A (2003) DNA height in atomic force microscopy. Ultramicroscopy 96:167–174PubMedCrossRefGoogle Scholar
  59. 59.
    Santos S, Thomson NH (2011) Energy dissipation in a dynamic nanoscale contact. Appl Phys Lett 98:013101–013103CrossRefGoogle Scholar
  60. 60.
    Crampton N, Bonass WA, Kirkham J, Rivetti C, Thomson NH (2006) Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy. Nucleic Acids Res 34:5416–5425PubMedCrossRefGoogle Scholar
  61. 61.
    Liu LF, Wang JC (1987) Supercoiling of the DNA-template during transcription. Proc Natl Acad Sci 84:7024–7027PubMedCrossRefGoogle Scholar
  62. 62.
    Beaglehole D, Christenson HK (1992) Vapor adsorption on mica and silicon: entropy effects, layering, and surface forces. J Phys Chem 96:3395–3403CrossRefGoogle Scholar
  63. 63.
    Balmer TE, Christenson HK, Spencer ND, Heuberger M (2008) The effect of surface ions on water adsorption to mica. Langmuir 24:1566–1569PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Sergio Santos
    • 1
    • 2
    • 3
  • Daniel Billingsley
    • 1
    • 2
  • Neil Thomson
    • 1
    • 2
  1. 1.Department of Oral BiologyUniversity of LeedsLeedsUK
  2. 2.School of Physics and AstronomyUniversity of LeedsLeedsUK
  3. 3.Laboratory for Energy and NanosciencesMasdar Institute of Science and TechnologyAbu DhabiUAE

Personalised recommendations