Biophysical Reviews

, Volume 10, Issue 2, pp 285–292 | Cite as

High-speed atomic force microscopy and its future prospects

  • Toshio Ando


Various techniques have been developed and used to investigate how proteins produce complex biological architectures and phenomena. Among these techniques, high-speed atomic force microscopy (HS-AFM) holds a unique position. It is only HS-AFM that allows the simultaneous assessment of structure and dynamics of single protein molecules in action. This new microscopy tool has been successfully applied to a variety of proteins, from motor proteins to membrane proteins, antibodies, enzymes, and even to intrinsically disordered proteins. And yet there still remain many biomolecular phenomena that cannot be addressed by HS-AFM in its current form. Here, I present a brief history of HS-AFM development, describe the current state of HS-AFM, and then discuss which new biological scanning probe microscopy techniques will be coming up next.


Imaging High-speed AFM Proteins Dynamic processes Structural changes 



This work was supported by JST/CREST (#JPMJCR13M1) and KAKENHI from the Ministry of Education, Culture, Sports, Science and Technology, Japan (#21113002, #24227005 and #26119003).

Compliance with ethical standards

Conflicts of interest

Toshio Ando declares that he has no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by the author.


  1. Ando T (2017a) Directly watching biomolecules in action by high-speed atomic force microscopy. Biophys Rev 9:421–429CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ando T (2017b) Technical development of high-speed AFM and its future prospects. Oyo Butsuri 86:867–874Google Scholar
  3. Ando T, Kodera N, Takai E, Maruyama D, Saito K, Toda A (2001) A high-speed atomic force microscope for studying biological macromolecules. Proc Natl Acad Sci USA 98:12468–12472CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ando T, Uchihashi T, Fukuma T (2008) High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes. Prog Surf Sci 83:337–437CrossRefGoogle Scholar
  5. Ando T, Uchihashi T, Scheuring S (2014) Filming biomolecular processes by high-speed atomic force microscopy. Chem Rev 114:3120–3188CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bezanilla M, Drake B, Nudler E, Kashlev M, Hansma PK, Hansma HG (1994) Motion and enzymatic degradation of DNA in the atomic force microscope. Biophys J 67:2454–2459CrossRefPubMedPubMedCentralGoogle Scholar
  7. Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56:930–933CrossRefPubMedGoogle Scholar
  8. Colom A, Casuso I, Boudier T, Scheuring S (2012) High-speed atomic force microscopy: cooperative adhesion and dynamic equilibrium of junctional microdomain membrane proteins. J Mol Biol 423:249–256CrossRefPubMedGoogle Scholar
  9. Colom A, Casuso I, Rico F, Scheuring S (2013) A hybrid high-speed atomic force–optical microscope for visualizing single membrane proteins on eukaryotic cells. Nat Commun 4:2155 (8 pp)CrossRefPubMedGoogle Scholar
  10. Drake B, Prater CB, Weisenhorn AL, Gould SA, Albrecht TR, Quate CF, Cannell DS, Hansma HG, Hansma PK (1989) Imaging crystals, polymers, and processes in water with the atomic force microscope. Science 243:1586–1589CrossRefPubMedGoogle Scholar
  11. Dufrene YF, Evans E, Engel A, Helenius J, Gaub HE, Müller DJ (2011) Five challenges to bringing single-molecule force spectroscopy into the living cell. Nat Methods 8:123–127CrossRefPubMedGoogle Scholar
  12. Düttmann M, Togashi Y, Yanagida T, Mikhailov AS (2012) Myosin-V as a mechanical sensor: an elastic network study. Biophys J 102:542–551CrossRefPubMedPubMedCentralGoogle Scholar
  13. Eggeling C, Willig KI, Sahl SJ, Hell SW (2015) Lens-based fluorescence nanoscopy. Q Rev Biophys 48:178–243CrossRefPubMedGoogle Scholar
  14. Erie DA, Yang G, Schultz HC, Bustamante C (1994) DNA bending by Cro protein in specific and nonspecific complexes: implications for protein site recognition and specificity. Science 266:1562–1566CrossRefPubMedGoogle Scholar
  15. Fukuda S, Uchihashi T, Iino R, Okazaki Y, Yoshida M, Igarashi K, Ando T (2013) High-speed atomic force microscope combined with single-molecule fluorescence microscope. Rev Sci Instrum 84:073706CrossRefPubMedGoogle Scholar
  16. Geng J, Kim K, Zhang J, Escalada A, Tunuguntla R, Comolli LR, Allen FI, Shnyrova AV, Cho KR, Munoz D, Wang YM, Grigoropoulos CP, Ajo-Franklin CM, Frolov VA, Noy A (2014) Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes. Nature 514:612–615CrossRefPubMedGoogle Scholar
  17. Guillaume-Gentil O, Potthoff E, Ossola D, Franz CM, Zambelli T, Vorholt JA (2014) Force-controlled manipulation of single cells: from AFM to FluidFM. Trend Biotechnol 32:381–388CrossRefGoogle Scholar
  18. Häberle W, Höber JKH, Ohnesorge F, Smith DPE, Binnig G (1992) In situ investigations of single living cells infected by viruses. Ultramicroscopy 42–44:1161–1167CrossRefGoogle Scholar
  19. Hansma PK, Drake B, Marti O, Gould SA, Prater CB (1989) The scanning ion-conductance microscope. Science 243:641–643CrossRefPubMedGoogle Scholar
  20. Hecht B, Sick B, Wild UP, Deckert V, Zenobi R, Martin OJF, Pohl DW (2000) Scanning near-field optical microscopy with aperture probes: fundamentals and applications. J Chem Phys 112:7761–7774CrossRefGoogle Scholar
  21. Kasas S, Thomson NH, Smith BL, Hansma HG, Zhu X, Guthold M, Bustamante C, Kool ET, Kashlev M, Hansma PK (1997) Escherichia Coli RNA polymerase activity observed using atomic force microscopy. Biochemistry 36:461–468CrossRefPubMedGoogle Scholar
  22. Kodera N, Sakashita M, Ando T (2006) Dynamic proportional-integral-differential controller for high-speed atomic force microscopy. Rev Sci Instrum 77:083704 (7 pp)CrossRefGoogle Scholar
  23. Kodera N, Yamashita H, Ando T (2005) Active damping of the scanner for high-speed atomic force microscopy. Rev Sci Instrum 76:053708 (5 pp)CrossRefGoogle Scholar
  24. Lin JN, Drake B, Lea AS, Hansma PK, Andrade JD (1990) Direct observation of immunoglobulin adsorption dynamics using the atomic force microscope. Langmuir 6:509–511CrossRefGoogle Scholar
  25. Lopez CF, Nielsen SO, Moore PB, Klein ML (2004) Understanding nature's design for a nanosyringe. Proc Natl Acad Sci USA 101:4431–4434CrossRefPubMedPubMedCentralGoogle Scholar
  26. Meyer G, Amer NM (1988) Novel optical approach to atomic force microscopy. Appl Phys Lett 53:1045–1047CrossRefGoogle Scholar
  27. Miyagi A, Tsunaka Y, Uchihashi T, Mayanagi K, Hirose S, Morikawa K, Ando T (2008) Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy. Chem Phys Chem 9:1859–1866CrossRefPubMedGoogle Scholar
  28. Obataya I, Nakamura C, Han SW, Nakamura N, Miyake J (2005) Mechanical sensing of the penetration of various nanoneedles into a living cell using atomic force microscopy. Biosens Bioelectron 20:1652–1655CrossRefPubMedGoogle Scholar
  29. Oestreicher Z, Taoka A, Fukumori Y (2015) A comparison of the surface nanostructure from two different types of gram-negative cells: Escherichia coli and Rhodobacter sphaeroides. Micron 72:8–14CrossRefPubMedGoogle Scholar
  30. Rief M, Oesterhelt F, Heymann B, Gaub H (1997) Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science 275:1295–1297CrossRefPubMedGoogle Scholar
  31. Sakiyama Y, Mazur A, Kapinos LE, Lim RYH (2016) Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nat Nanotechnol 11:719–723CrossRefPubMedGoogle Scholar
  32. Schabert FA, Engel A (1994) Reproducible acquisition of Escherichia coli porin surface topographs by atomic force microscopy. Biophys J 67:2394–2403CrossRefPubMedPubMedCentralGoogle Scholar
  33. Shevchuk AI, Frolenkov GI, Sanchez D, James PS, Freedman N, Lab MJ, Jones R, Klenerman D, Korchev YE (2006) Imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy. Angew Chem Int Ed Engl 45:2212–2216CrossRefPubMedGoogle Scholar
  34. Shibata M, Uchihashi T, Ando T, Yasuda R (2015) Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells. Sci Rep 5:8724CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sanii B, Ashby PD (2010) High-sensitivity deflection detection of nanowires. Phys Rev Lett 104:147203CrossRefPubMedGoogle Scholar
  36. Togashi Y, Mikhailov AS (2007) Nonlinear relaxation dynamics in elastic networks and design principles of molecular machines. Proc Natl Acad Sci USA 104:8697–8702CrossRefPubMedPubMedCentralGoogle Scholar
  37. Uchihashi T, Ando T, Yamashita H (2006) Fast phase imaging in liquids using a rapid scan atomic force microscope. Appl Phys Lett 89:213112 (3 pp)CrossRefGoogle Scholar
  38. Uchihashi T, Iino R, Ando T, Noji H (2011) High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333:755–758CrossRefPubMedGoogle Scholar
  39. Viani MB, Schäffer TE, Paloczi GT, Pietrasanta LI, Smith BL, Thompson JB, Richter M, Rief M, Gaub HE, Plaxco KW, Cleland AN, Hansma HG, Hansma PK (1999) Fast imaging and fast force spectroscopy of single biopolymers with a new atomic force microscope designed for small cantilevers. Rev Sci Instrum 70:4300–4303CrossRefGoogle Scholar
  40. Viani MB, Pietrasanta LI, Thompson JB, Chand A, Gebeshuber IC, Kindt JH, Richter M, Hansma HG, Hansma PK (2000) Probing protein–protein interactions in real time. Nat Struct Biol 7:644–647CrossRefPubMedGoogle Scholar
  41. Wang RY-R, Kudryashev M, Li X, Egelman EH, Basler M, Cheng Y, Baker D, DiMaio F (2015) De novo protein structure determination from near-atomic-resolution cryo-EM maps. Nat Methods 12:335–338CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yamashita H, Taoka A, Uchihashi T, Asano T, Ando T, Fukumori Y (2012) Single molecule imaging on living bacterial cell surface by high-speed AFM. J Mol Biol 422:300–309CrossRefPubMedGoogle Scholar
  43. Zhong Q, Inniss D, Kjoller K, Elings VB (1993) Fractured polymer/scilica fiber surfaces studied by tapping mode atomic force microscopy. Surf Sci Lett 290:L688–L692Google Scholar

Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Nano-Life Science InstituteKanazawa UniversityKanazawaJapan
  2. 2.Core Research for Evolutionary Science and Technology (CREST) Japan Science and Technology Agency (JST)TokyoJapan

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