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Atomic Force Microscopy: An Introduction

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Single Molecule Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2694))

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Abstract

Imaging of nano-sized particles and sample features is crucial in a variety of research fields, for instance, in biological sciences, where it is paramount to investigate structures at the single particle level. Often, two-dimensional images are not sufficient, and further information such as topography and mechanical properties are required. Furthermore, to increase the biological relevance, it is desired to perform the imaging in close to physiological environments. Atomic force microscopy (AFM) meets these demands in an all-in-one instrument. It provides high-resolution images including surface height information leading to three-dimensional information on sample morphology. AFM can be operated both in air and in buffer solutions. Moreover, it has the capacity to determine protein and membrane material properties via the force spectroscopy mode. Here we discuss the principles of AFM operation and provide examples of how biomolecules can be studied. New developments in AFM are discussed, and by including approaches such as bimodal AFM and high-speed AFM (HS-AFM), we show how AFM can be used to study a variety of static and dynamic single biomolecules and biomolecular assemblies.

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References

  1. Demtröder W (2010) Experimentalphysik 3: Kern-, Teilchen-und Astrophysik, 4th edn. Springer, Berlin

    Book  MATH  Google Scholar 

  2. Binnig G, Rohrer H, Gerber C et al (1982) Surface studies by scanning tunneling microscopy. Phys Rev Lett 49(1):57

    Article  ADS  Google Scholar 

  3. Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56(9):930

    Article  ADS  Google Scholar 

  4. de Pablo PJ (2011) Introduction to atomic force microscopy. In: Method molecule biology, Humana Press, Totowa, NJ, Springer, 738:197–212

    Google Scholar 

  5. Voigtländer B (2019) Atomic force microscopy. Springer, Berlin

    Book  Google Scholar 

  6. Santos NC, Carvalho FA (2019) Atomic force microscopy. In: Meth Mol biol, vol 1886. Springer, Berlin

    Google Scholar 

  7. Kodera N, Ando T (2014) The path to visualization of walking myosin V by high-speed atomic force microscopy. Biophys Rev 6(3):237–260

    Article  Google Scholar 

  8. Marchetti M, Wuite G, Roos W (2016) Atomic force microscopy observation and characterization of single virions and virus-like particles by nano-indentation. Curr Opin Virol 18:82–88

    Article  Google Scholar 

  9. Krieg M, Fläschner G, Alsteens D et al (2019) Atomic force microscopy-based mechanobiology. Nat Rev Phys 1(1):41–57

    Article  Google Scholar 

  10. Morris VJ, Kirby AR, Gunning PA (2009) Atomic force microscopy for biologists, 2nd edn. Imperial College Press, London

    Book  Google Scholar 

  11. Vorselen D, van Dommelen SM, Sorkin R et al (2018) The fluid membrane determines mechanics of erythrocyte extracellular vesicles and is softened in hereditary spherocytosis. Nat Commun 9(1):1–9

    Article  Google Scholar 

  12. Maity S, Trinco G, Buzón P et al (2022) High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS. Proc Natl Acad Sci U S A 119(6):e2113927119

    Article  Google Scholar 

  13. Müller DJ, Dumitru AC, Lo Giudice C et al (2020) Atomic force microscopy-based force spectroscopy and multiparametric imaging of biomolecular and cellular systems. Chem Rev 121(19):11701–11725

    Article  Google Scholar 

  14. Bruinsma RF, Wuite GJ, Roos WH (2021) Physics of viral dynamics. Nat Rev Phys 3(2):76–91

    Article  Google Scholar 

  15. Eaton P, West P (2010) Atomic force microscopy. Oxford university press, Oxford

    Book  Google Scholar 

  16. Ando T (2012) High-speed atomic force microscopy coming of age. Nanotechnology 23(6):062001

    Article  ADS  Google Scholar 

  17. Uchihashi T, Scheuring S (2018) Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes. Biochim Biophys Acta Gen Subj 1862(2):229–240

    Article  Google Scholar 

  18. Ando T (2022) High-speed atomic force microscopy in biology: directly watching dynamics of biomolecules in action. Springer, Heidelberg

    Book  Google Scholar 

  19. Meyer G, Amer NM (1988) Novel optical approach to atomic force microscopy. Appl Phys Lett 53(12):1045–1047

    Article  ADS  Google Scholar 

  20. Churnside AB, Sullan RMA, Nguyen DM et al (2012) Routine and timely sub-picoNewton force stability and precision for biological applications of atomic force microscopy. Nano Lett 12(7):3557–3561

    Article  ADS  Google Scholar 

  21. Sader JE, Chon JW, Mulvaney P (1999) Calibration of rectangular atomic force microscope cantilevers. Rev Sci Instrum 70(10):3967–3969

    Article  ADS  Google Scholar 

  22. Vorselen D, Kooreman ES, Wuite GJ et al (2016) Controlled tip wear on high roughness surfaces yields gradual broadening and rounding of cantilever tips. Sci Rep 6(1):1–7

    Article  Google Scholar 

  23. Heath GR, Kots E, Robertson JL et al (2021) Localization atomic force microscopy. Nature 594(7863):385–390

    Article  ADS  Google Scholar 

  24. Hölscher H, Allers W, Schwarz U et al (2000) Interpretation of “true atomic resolution” images of graphite (0001) in noncontact atomic force microscopy. Phys Rev B 62(11):6967

    Article  ADS  Google Scholar 

  25. Ho H, West P (1996) Optimizing AC-mode atomic force microscope imaging. J Scan Microsc 18(5):339–343

    Google Scholar 

  26. Garcıa R, Perez R (2002) Dynamic atomic force microscopy methods. Surf Sci Rep 47(6–8):197–301

    Article  ADS  Google Scholar 

  27. Garcia R, Herruzo ET (2012) The emergence of multifrequency force microscopy. Nat Nanotechnol 7(4):217–226

    Article  ADS  Google Scholar 

  28. Martínez NF, García R (2006) Measuring phase shifts and energy dissipation with amplitude modulation atomic force microscopy. Nanotechnology 17(7):S167

    Article  ADS  Google Scholar 

  29. Rodrıguez TR, Garcı́a R (2004) Compositional mapping of surfaces in atomic force microscopy by excitation of the second normal mode of the microcantilever. Appl Phys Lett 84(3):449–451

    Article  ADS  Google Scholar 

  30. Martinez N, Lozano JR, Herruzo E et al (2008) Bimodal atomic force microscopy imaging of isolated antibodies in air and liquids. Nanotechnology 19(38):384011

    Article  Google Scholar 

  31. Martínez-Martín D, Herruzo ET, Dietz C et al (2011) Noninvasive protein structural flexibility mapping by bimodal dynamic force microscopy. Phys Rev Lett 106(19):198101

    Article  ADS  Google Scholar 

  32. Patil S, Martinez NF, Lozano JR et al (2007) Force microscopy imaging of individual protein molecules with sub-pico Newton force sensitivity. J Mol Recognit 20(6):516–523

    Article  Google Scholar 

  33. De Pablo P, Colchero J, Gomez-Herrero J et al (1998) Jumping mode scanning force microscopy. Appl Phys Lett 73(22):3300–3302

    Article  ADS  Google Scholar 

  34. Moreno-Herrero F, Colchero J, Gomez-Herrero J et al (2004) Atomic force microscopy contact, tapping, and jumping modes for imaging biological samples in liquids. Phys Rev E 69(3):031915

    Article  ADS  Google Scholar 

  35. JPK Instruments (2011) Nanowizard 4–the next benchmark for BioAFM. JPK Instruments, Berlin

    Google Scholar 

  36. Bruker (2015) Peak-force tapping – how AFM should be. Bruker Nano Surfaces Division, Goleta

    Google Scholar 

  37. Zemła J, Danilkiewicz J, Orzechowska B et al (2018) Atomic force microscopy as a tool for assessing the cellular elasticity and adhesiveness to identify cancer cells and tissues. Semin Cell Dev Biol 73:115–124

    Article  Google Scholar 

  38. Scholl ZN, Li Q, Josephs E et al (2019) Force spectroscopy of single protein molecules using an atomic force microscope. JoVE (144):e55989

    Google Scholar 

  39. De Pablo P, Colchero J, Gomez-Herrero J et al (1999) Adhesion maps using scanning force microscopy techniques. J Adhes 71(4):339–356

    Article  Google Scholar 

  40. Viljoen A, Mathelié-Guinlet M, Ray A et al (2021) Force spectroscopy of single cells using atomic force microscopy. Nat Rev Methods Primers 1(1):1–24

    Article  Google Scholar 

  41. Mitsui K, Hara M, Ikai A (1996) Mechanical unfolding of a2-macroglobulin molecules with atomic force microscope. FEBS Lett 385(1–2):29–33

    Article  Google Scholar 

  42. Rief M, Gautel M, Oesterhelt F et al (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276(5315):1109–1112

    Article  Google Scholar 

  43. Maity S, Lyubchenko YL (2019) Force clamp approach for characterization of nano-assembly in amyloid beta 42 dimer. Nanoscale 11(25):12259–12265

    Article  Google Scholar 

  44. Mignolet J, Mathelié-Guinlet M, Viljoen A et al (2021) AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction. Nanoscale Horiz 6(6):489–496

    Article  ADS  Google Scholar 

  45. Wang Y-F, Zhang Q, Tian F et al (2022) Spatiotemporal tracing of the cellular internalization process of rod-shaped nanostructures. ACS Nano 16(3):4059–4071

    Article  Google Scholar 

  46. Medalsy I, Hensen U, Muller DJ (2011) Imaging and quantifying chemical and physical properties of native proteins at molecular resolution by force–volume AFM. Angew Chem Int Ed 50(50):12103–12108

    Article  Google Scholar 

  47. Yang Y, Xiao X, Peng Y et al (2019) The comparison between force volume and peak force quantitative nanomechanical mode of atomic force microscope in detecting cell’s mechanical properties. Microsc Res Tech 82(11):1843–1851

    Google Scholar 

  48. Penedo M, Miyazawa K, Okano N et al (2021) Visualizing intracellular nanostructures of living cells by nanoendoscopy-AFM. Sci Adv 7(52):eabj4990

    Article  ADS  Google Scholar 

  49. Baclayon M, Wuite G, Roos W (2010) Imaging and manipulation of single viruses by atomic force microscopy. Soft Matter 6(21):5273–5285

    Article  ADS  Google Scholar 

  50. Bustamante C, Rivetti C (1996) Visualizing protein-nucleic acid interactions on a large scale with the scanning force microscope. Annu Rev Biophys Biomol Struct 25(1):395–429

    Article  Google Scholar 

  51. Farge G, Mehmedovic M, Baclayon M et al (2014) In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription. Cell Rep 8(1):66–74

    Article  Google Scholar 

  52. Sanchez H, Kanaar R, Wyman C (2010) Molecular recognition of DNA–protein complexes: a straightforward method combining scanning force and fluorescence microscopy. Ultramicroscopy 110(7):844–851

    Article  Google Scholar 

  53. Falvo M, Washburn S, Superfine R et al (1997) Manipulation of individual viruses: friction and mechanical properties. Biophys J 72(3):1396–1403

    Article  Google Scholar 

  54. van der Heijden T, Moreno-Herrero F, Kanaar R et al (2006) Comment on “Direct and real-time visualization of the disassembly of a single RecA-DNA-ATPγS complex using AFM imaging in fluid”. Nano Lett 6(12):3000–3002

    Article  ADS  Google Scholar 

  55. Ando T (2018) High-speed atomic force microscopy and its future prospects. Biophys Rev 10(2):285–292

    Article  Google Scholar 

  56. Ando T, Kodera N, Takai E et al (2001) A high-speed atomic force microscope for studying biological macromolecules. Proc Natl Acad Sci U S A 98(22):12468–12472

    Article  ADS  Google Scholar 

  57. Ando T, Uchihashi T, Kodera N et al (2008) High-speed AFM and nano-visualization of biomolecular processes. Pflug Arch Eur J Physiol 456(1):211–225

    Article  Google Scholar 

  58. Kodera N, Yamamoto D, Ishikawa R et al (2010) Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468(7320):72–76

    Article  ADS  Google Scholar 

  59. Ando T, Uchihashi T, Fukuma T (2008) High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes. Prog Surf Sci 83(7–9):337–437

    Article  ADS  Google Scholar 

  60. Miyagi A, Scheuring S (2018) A novel phase-shift-based amplitude detector for a high-speed atomic force microscope. Rev Sci Instrum 89(8):083704

    Article  ADS  Google Scholar 

  61. Yang C, Yan J, Dukic M et al (2016) Design of a high-bandwidth tripod scanner for high speed atomic force microscopy. Scanning 38(6):889–900

    Article  Google Scholar 

  62. Fukuda S, Ando T (2021) Faster high-speed atomic force microscopy for imaging of biomolecular processes. Rev Sci Instrum 92(3):033705

    Article  ADS  Google Scholar 

  63. Matin TR, Heath GR, Huysmans GH et al (2020) Millisecond dynamics of an unlabeled amino acid transporter. Nat Commun 11(1):1–11

    Article  Google Scholar 

  64. Perrino AP, Miyagi A, Scheuring S (2021) Single molecule kinetics of bacteriorhodopsin by HS-AFM. Nat Commun 12(1):1–10

    Article  Google Scholar 

  65. Uchihashi T, Watanabe H, Fukuda S et al (2016) Functional extension of high-speed AFM for wider biological applications. Ultramicroscopy 160:182–196

    Article  Google Scholar 

  66. Valbuena A, Maity S, Mateu MG et al (2020) Visualization of single molecules building a viral capsid protein lattice through stochastic pathways. ACS Nano 14(7):8724–8734

    Article  Google Scholar 

  67. Gisbert VG, Benaglia S, Uhlig MR et al (2021) High-speed nanomechanical mapping of the early stages of collagen growth by bimodal force microscopy. ACS Nano 15(1):1850–1857

    Article  Google Scholar 

  68. Muller DJ (2008) AFM: a nanotool in membrane biology. Biochemistry 47(31):7986–7998

    Article  Google Scholar 

  69. Lamolle SF, Monjo M, Lyngstadaas SP et al (2009) Titanium implant surface modification by cathodic reduction in hydrofluoric acid: surface characterization and in vivo performance. J Biomed Mater Res A 88(3):581–588

    Article  Google Scholar 

  70. Larsson Wexell C, Thomsen P, Aronsson B-O et al (2013) Bone response to surface-modified titanium implants: studies on the early tissue response to implants with different surface characteristics. Int J Biomater 2013:412482

    Article  Google Scholar 

  71. Kroeze R, Helder M, Roos W et al (2010) The effect of ethylene oxide, glow discharge and electron beam on the surface characteristics of poly (L-lactide-co-caprolactone) and the corresponding cellular response of adipose stem cells. Acta Biomater 6(6):2060–2065

    Article  Google Scholar 

  72. Alsteens D, Gaub HE, Newton R et al (2017) Atomic force microscopy-based characterization and design of biointerfaces. Nat Rev Mater 2(5):1–16

    Article  Google Scholar 

  73. Baclayon M, Pv U, Mouhib H et al (2016) Mechanical unfolding of an autotransporter passenger protein reveals the secretion starting point and processive transport intermediates. ACS Nano 10(6):5710–5719

    Article  Google Scholar 

  74. Delguste M, Zeippen C, Machiels B et al (2018) Multivalent binding of herpesvirus to living cells is tightly regulated during infection. Sci Adv 4(8):eaat1273

    Article  ADS  Google Scholar 

  75. Maity S, Mazzolini M, Arcangeletti M et al (2015) Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy. Nat Commun 6(1):1–16

    Article  Google Scholar 

  76. Koehler M, Lo Giudice C, Vogl P et al (2020) Control of ligand-binding specificity using photocleavable linkers in AFM force spectroscopy. Nano Lett 20(5):4038–4042

    Article  ADS  Google Scholar 

  77. Buzón P, Maity S, Roos WH (2020) Physical virology: from virus self-assembly to particle mechanics. Wiley Interdiscip Rev Nanomed Nanobiotechnol 12(4):e1613

    Article  Google Scholar 

  78. Mateu MG (2012) Mechanical properties of viruses analyzed by atomic force microscopy: a virological perspective. Virus Res 168(1–2):1–22

    Article  ADS  Google Scholar 

  79. Roos W, Bruinsma R, Wuite G (2010) Physical virology. Nat Phys 6(10):733–743

    Article  Google Scholar 

  80. Vorselen D, Piontek MC, Roos WH et al (2020) Mechanical characterization of liposomes and extracellular vesicles, a protocol. Front Mol Biosci 7:139

    Article  Google Scholar 

  81. Roos WH, Gertsman I, May ER et al (2012) Mechanics of bacteriophage maturation. Proc Natl Acad Sci U S A 109(7):2342–2347

    Article  ADS  Google Scholar 

  82. Carrasco C, Luque A, Hernando-Pérez M et al (2011) Built-in mechanical stress in viral shells. Biophys J 100(4):1100–1108

    Article  Google Scholar 

  83. Baclayon M, Shoemaker GK, Uetrecht C et al (2011) Prestress strengthens the shell of Norwalk virus nanoparticles. Nano Lett 11(11):4865–4869

    Article  ADS  Google Scholar 

  84. Denning D, Bennett S, Mullen T et al (2019) Maturation of adenovirus primes the protein nano-shell for successful endosomal escape. Nanoscale 11(9):4015–4024

    Article  Google Scholar 

  85. Snijder J, Ivanovska I, Baclayon M et al (2012) Probing the impact of loading rate on the mechanical properties of viral nanoparticles. Micron 43(12):1343–1350

    Article  Google Scholar 

  86. Rico F, Russek A, Gonzalez L et al (2019) Heterogeneous and rate-dependent streptavidin–biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations. Proc Natl Acad Sci U S A 116(14):6594–6601

    Article  ADS  Google Scholar 

  87. Roos WH, Radtke K, Kniesmeijer E et al (2009) Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids. Proc Natl Acad Sci U S A 106(24):9673–9678

    Article  ADS  Google Scholar 

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Acknowledgment

This work is supported by the EU FET Proactive ViruScan program. We thank Melissa Piontek for her work on the previous version of this chapter, on which this chapter is based.

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Authors and Affiliations

  1. Moleculaire Biofysica, Zernike instituut, Rijksuniversiteit Groningen, Groningen, the Netherlands

    Yuzhen Feng & Wouter H. Roos

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  1. Yuzhen Feng
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  2. Wouter H. Roos
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Correspondence to Wouter H. Roos .

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Editors and Affiliations

  1. LaserLaB Amsterdam and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

    Iddo Heller

  2. LaserLaB Amsterdam and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

    David Dulin

  3. LaserLaB Amsterdam and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

    Erwin J.G. Peterman

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Feng, Y., Roos, W.H. (2024). Atomic Force Microscopy: An Introduction. In: Heller, I., Dulin, D., Peterman, E.J. (eds) Single Molecule Analysis . Methods in Molecular Biology, vol 2694. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3377-9_14

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  • DOI: https://doi.org/10.1007/978-1-0716-3377-9_14

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