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  • Review Article
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Atomic force microscopy-based characterization and design of biointerfaces

Abstract

Atomic force microscopy (AFM)-based methods have matured into a powerful nanoscopic platform, enabling the characterization of a wide range of biological and synthetic biointerfaces ranging from tissues, cells, membranes, proteins, nucleic acids and functional materials. Although the unprecedented signal-to-noise ratio of AFM enables the imaging of biological interfaces from the cellular to the molecular scale, AFM-based force spectroscopy allows their mechanical, chemical, conductive or electrostatic, and biological properties to be probed. The combination of AFM-based imaging and spectroscopy structurally maps these properties and allows their 3D manipulation with molecular precision. In this Review, we survey basic and advanced AFM-related approaches and evaluate their unique advantages and limitations in imaging, sensing, parameterizing and designing biointerfaces. It is anticipated that in the next decade these AFM-related techniques will have a profound influence on the way researchers view, characterize and construct biointerfaces, thereby helping to solve and address fundamental challenges that cannot be addressed with other techniques.

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Figure 1: AFM imaging principles and applications characterizing biointerfaces.
Figure 2: AFM-based force spectroscopy from single molecules to cells.
Figure 3: AFM-based imaging and mapping of mechanical properties of biointerfaces.
Figure 4: AFM-based imaging and affinity mapping of biointerfaces.
Figure 5: Characterizing reactions of biointerfaces in real time using AFM-based microsensors.
Figure 6: AFM-based sculpting, patterning and assembly of biointerfaces.
Figure 7: Combining AFM with other microscopic and spectroscopic approaches.

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References

  1. Nel, A. E. et al. Understanding biophysicochemical interactions at the nano–bio interface. Nat. Mater. 8, 543–557 (2009).

    Article  CAS  Google Scholar 

  2. Ross, A. M. & Lahann, J. Current trends and challenges in biointerfaces science and engineering. Annu. Rev. Chem. Biomol. 6, 161–186 (2015).

    Article  CAS  Google Scholar 

  3. Stevens, M. M. & George, J. H. Exploring and engineering the cell surface interface. Science 310, 1135–1138 (2005).

    Article  CAS  Google Scholar 

  4. Andrews, R. N., Co, C. C. & Ho, C. C. Engineering dynamic biointerfaces. Curr. Opin. Chem. Eng. 11, 28–33 (2016).

    Article  Google Scholar 

  5. Gerber, C. & Lang, H. P. How the doors to the nanoworld were opened. Nat. Nanotechnol. 1, 3–5 (2006).

    Article  CAS  Google Scholar 

  6. Binnig, G., Quate, C. F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986). This paper describes the invention of AFM.

    Article  CAS  Google Scholar 

  7. Drake, B. et al. Imaging crystals, polymers, and processes in water with the atomic force microscope. Science 243, 1586–1589 (1989).

    Article  CAS  Google Scholar 

  8. Radmacher, M., Tillmann, R. W., Fritz, M. & Gaub, H. E. From molecules to cells: imaging soft samples with the atomic force microscope. Science 257, 1900–1905 (1992).

    Article  CAS  Google Scholar 

  9. Garcia, R. & Herruzo, E. T. The emergence of multifrequency force microscopy. Nat. Nanotechnol. 7, 217–226 (2012).

    Article  CAS  Google Scholar 

  10. Ando, T., Uchihashi, T. & Kodera, N. High-speed AFM and applications to biomolecular systems. Ann. Rev. Biophys. 42, 393–414 (2013).

    Article  CAS  Google Scholar 

  11. Zhang, S., Aslan, H., Besenbacher, F. & Dong, M. Quantitative biomolecular imaging by dynamic nanomechanical mapping. Chem. Soc. Rev. 43, 7412–7429 (2014).

    Article  CAS  Google Scholar 

  12. Dufrêne, Y. F. et al. Imaging modes of atomic force microscopy for application in molecular and cell biology. Nat. Nanotechnol. http://dx.doi.org/10.1038/nnano.2017.45 (2017).

  13. Frisbie, C. D., Rozsnyai, L. F., Noy, A., Wrighton, M. S. & Lieber, C. M. Functional group imaging by chemical force microscopy. Science 265, 2071–2074 (1994).

    Article  CAS  Google Scholar 

  14. Hinterdorfer, P. & Dufrêne, Y. F. Detection and localization of single molecular recognition events using atomic force microscopy. Nat. Methods 3, 347–355 (2006).

    Article  CAS  Google Scholar 

  15. Müller, D. J., Helenius, J., Alsteens, D. & Dufrêne, Y. F. Force probing surfaces of living cells to molecular resolution. Nat. Chem. Biol. 5, 383–390 (2009).

    Article  CAS  Google Scholar 

  16. Dufrêne, Y. F., Martinez-Martin, D., Medalsy, I., Alsteens, D. & Müller, D. J. Multiparametric imaging of biological systems by force–distance curve-based AFM. Nat. Methods 10, 847–854 (2013).

    Article  CAS  Google Scholar 

  17. Lang, H. P. & Gerber, C. Microcantilever sensors. Top. Curr. Chem. 285, 1–27 (2008).

    Article  CAS  Google Scholar 

  18. Müller, D. J. & Dufrêne, Y. F. Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat. Nanotechnol. 3, 261–269 (2008).

    Article  CAS  Google Scholar 

  19. Pires, D. et al. Nanoscale three-dimensional patterning of molecular resists by scanning probes. Science 328, 732–735 (2010).

    Article  CAS  Google Scholar 

  20. Garcia, R., Knoll, A. W. & Riedo, E. Advanced scanning probe lithography. Nat. Nanotechnol. 9, 577–587 (2014).

    Article  CAS  Google Scholar 

  21. Puchner, E. M. & Gaub, H. E. Single-molecule mechanoenzymatics. Ann. Rev. Biophys. 41, 497–518 (2012).

    Article  CAS  Google Scholar 

  22. Cattin, C. J. et al. Mechanical control of mitotic progression in single animal cells. Proc. Natl Acad. Sci. USA 112, 11258–11263 (2015).

    Article  CAS  Google Scholar 

  23. Kufer, S. K., Puchner, E. M., Gumpp, H., Liedl, T. & Gaub, H. E. Single-molecule cut-and-paste surface assembly. Science 319, 594–596 (2008).

    Article  CAS  Google Scholar 

  24. Engel, A. & Müller, D. J. Observing single biomolecules at work with the atomic force microscope. Nat. Struct. Biol. 7, 715–718 (2000).

    Article  CAS  Google Scholar 

  25. Kuznetsov, Y. G. & McPherson, A. Atomic force microscopy in imaging of viruses and virus-infected cells. Microbiol. Mol. Biol. Rev. 75, 268–285 (2011).

    Article  CAS  Google Scholar 

  26. Ido, S. et al. Beyond the helix pitch: direct visualization of native DNA in aqueous solution. ACS Nano 7, 1817–1822 (2013).

    Article  CAS  Google Scholar 

  27. Pyne, A., Thompson, R., Leung, C., Roy, D. & Hoogenboom, B. W. Single-molecule reconstruction of oligonucleotide secondary structure by atomic force microscopy. Small 10, 3257–3261 (2014).

    Article  CAS  Google Scholar 

  28. Ido, S. et al. Immunoactive two-dimensional self-assembly of monoclonal antibodies in aqueous solution revealed by atomic force microscopy. Nat. Mater. 13, 264–270 (2014).

    Article  CAS  Google Scholar 

  29. Seelert, H. et al. Proton powered turbine of a plant motor. Nature 405, 418–419 (2000).

    Article  CAS  Google Scholar 

  30. Fotiadis, D. et al. Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 421, 127–128 (2003).

    Article  CAS  Google Scholar 

  31. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333, 755–758 (2011).

    Article  CAS  Google Scholar 

  32. Müller, D. J., Hand, G. M., Engel, A. & Sosinsky, G. Conformational changes in surface structures of isolated Connexin26 gap junctions. EMBO J. 21, 3598–3607 (2002).

    Article  Google Scholar 

  33. Müller, D. J. & Engel, A. Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy. J. Mol. Biol. 285, 1347–1351 (1999).

    Article  Google Scholar 

  34. Mari, S. A. et al. pH-induced conformational change of the beta-barrel-forming protein OmpG reconstituted into native E. coli lipids. J. Mol. Biol. 396, 610–616 (2010).

    Article  CAS  Google Scholar 

  35. Czajkowsky, D. M., Hotze, E. M., Shao, Z. & Tweten, R. K. Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane. EMBO J. 23, 3206–3215 (2004).

    Article  CAS  Google Scholar 

  36. Shibata, M., Uchihashi, T., Yamashita, H., Kandori, H. & Ando, T. Structural changes in bacteriorhodopsin in response to alternate illumination observed by high-speed atomic force microscopy. Angew. Chem. Int. Ed. 50, 4410–4413 (2011).

    Article  CAS  Google Scholar 

  37. Mari, S. A. et al. Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains. Proc. Natl Acad. Sci. USA 108, 20802–20807 (2011).

    Article  Google Scholar 

  38. Rangl, M. et al. Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels. Nat. Commun. 7, 12789 (2016).

    Article  CAS  Google Scholar 

  39. Müller, D. J. et al. Observing membrane protein diffusion at subnanometer resolution. J. Mol. Biol. 327, 925–930 (2003).

    Article  CAS  Google Scholar 

  40. Karner, A. et al. Tuning membrane protein mobility by confinement into nanodomains. Nat. Nanotechnol. http://dx.doi.org/10.1038/nnano.2016.236 (2016).

  41. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010). This study describes single-motor proteins walking along actin filaments.

    Article  CAS  Google Scholar 

  42. Cisneros, D. A., Hung, C., Franz, C. M. & Müller, D. J. Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy. J. Struct. Biol. 154, 232–245 (2006).

    Article  CAS  Google Scholar 

  43. Stamov, D. R., Stock, E., Franz, C. M., Jahnke, T. & Haschke, H. Imaging collagen type I fibrillogenesis with high spatiotemporal resolution. Ultramicroscopy 149, 86–94 (2015).

    Article  CAS  Google Scholar 

  44. Lehto, T., Miaczynska, M., Zerial, M., Müller, D. J. & Severin, F. Observing the growth of individual actin filaments in cell extracts by time-lapse atomic force microscopy. FEBS Lett. 551, 25–28 (2003).

    Article  CAS  Google Scholar 

  45. Sharma, S. et al. Nanostructured self-assembly of inverted formin 2 (INF2) and F-actin-INF2 complexes revealed by atomic force microscopy. Langmuir 30, 7533–7539 (2014).

    Article  CAS  Google Scholar 

  46. Friedrichs, J., Taubenberger, A., Franz, C. M. & Müller, D. J. Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM. J. Mol. Biol. 372, 594–607 (2007).

    Article  CAS  Google Scholar 

  47. Gudzenko, T. & Franz, C. M. Studying early stages of fibronectin fibrillogenesis in living cells by atomic force microscopy. Mol. Biol. Cell 26, 3190–3204 (2015).

    Article  CAS  Google Scholar 

  48. Stark, M., Stark, R. W., Heckl, W. M. & Guckenberger, R. Inverting dynamic force microscopy: from signals to time-resolved interaction forces. Proc. Natl Acad. Sci. USA 99, 8473–8478 (2002).

    Article  CAS  Google Scholar 

  49. Martinez-Martin, D., Herruzo, E. T., Dietz, C., Gomez-Herrero, J. & Garcia, R. Noninvasive protein structural flexibility mapping by bimodal dynamic force microscopy. Phys. Rev. Lett. 106, 198101 (2011).

    Article  CAS  Google Scholar 

  50. Raman, A. et al. Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy. Nat. Nanotechnol. 6, 809–814 (2011).

    Article  CAS  Google Scholar 

  51. Hansma, P. K., Schitter, G., Fantner, G. E. & Prater, C. High-speed atomic force microscopy. Science 314, 601–602 (2006).

    Article  CAS  Google Scholar 

  52. Fantner, G. E. et al. Components for high speed atomic force microscopy. Ultramicroscopy 106, 881–887 (2006).

    Article  CAS  Google Scholar 

  53. Viani, M. B. et al. Probing protein–protein interactions in real time. Nat. Struct. Biol. 7, 644–647 (2000). This paper introduces the use of ultrashort AFM cantilevers to detect fast interactions, which is the basis for high-speed AFM imaging and force spectroscopy.

    Article  CAS  Google Scholar 

  54. Watanabe-Nakayama, T., Itami, M., Kodera, N., Ando, T. & Konno, H. High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils. Sci. Rep. 6, 28975 (2016).

    Article  CAS  Google Scholar 

  55. Chiaruttini, N. et al. Relaxation of loaded ESCRT-III spiral springs drives membrane deformation. Cell 163, 866–879 (2015).

    Article  CAS  Google Scholar 

  56. Yamashita, H. et al. Single-molecule imaging on living bacterial cell surface by high-speed AFM. J. Mol. Biol. 422, 300–309 (2012).

    Article  CAS  Google Scholar 

  57. Sakiyama, Y., Mazur, A., Kapinos, L. E. & Lim, R. Y. Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nat. Nanotechnol. 11, 719–723 (2016).

    Article  CAS  Google Scholar 

  58. Butt, H. J., Cappella, B. & Kappl, M. Force measurements with the atomic force microscope: technique, interpretation and applications. Surf. Sci. Rep. 59, 1–152 (2005).

    Article  CAS  Google Scholar 

  59. Ducker, W. A., Senden, T. J. & Pashley, R. M. Direct measurement of colloidal forces using an atomic force microscope. Nature 353, 239–241 (1991).

    Article  CAS  Google Scholar 

  60. Pelling, A. E., Sehati, S., Gralla, E. B., Valentine, J. S. & Gimzewski, J. K. Local nanomechanical motion of the cell wall of Saccharomyces cerevisiae. Science 305, 1147–1150 (2004).

    Article  CAS  Google Scholar 

  61. Krieg, M., Dunn, A. R. & Goodman, M. B. Mechanical control of the sense of touch by beta-spectrin. Nat. Cell Biol. 16, 224–233 (2014).

    Article  CAS  Google Scholar 

  62. Vasquez, V., Krieg, M., Lockhead, D. & Goodman, M. B. Phospholipids that contain polyunsaturated fatty acids enhance neuronal cell mechanics and touch sensation. Cell Rep. 6, 70–80 (2014).

    Article  CAS  Google Scholar 

  63. Krieg, M. et al. Tensile forces govern germ-layer organization in zebrafish. Nat. Cell Biol. 10, 429–436 (2008). This study uses AFM to settle a long-standing dispute regarding whether it is cell adhesion or cortex tension that is responsible for cell sorting in the developing zebrafish embyro.

    Article  CAS  Google Scholar 

  64. Strilic, B. et al. Electrostatic cell-surface repulsion initiates lumen formation in developing blood vessels. Curr. Biol. 20, 2003–2009 (2010).

    Article  CAS  Google Scholar 

  65. Matzke, R., Jacobson, K. & Radmacher, M. Direct, high-resolution measurement of furrow stiffening during division of adherent cells. Nat. Cell Biol. 3, 607–610 (2001).

    Article  CAS  Google Scholar 

  66. Stewart, M. P. et al. Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature 469, 226–230 (2011).

    Article  CAS  Google Scholar 

  67. Cross, S. E., Jin, Y. S., Rao, J. & Gimzewski, J. K. Nanomechanical analysis of cells from cancer patients. Nat. Nanotechnol. 2, 780–783 (2007).

    Article  CAS  Google Scholar 

  68. Iyer, S., Gaikwad, R. M., Subba-Rao, V., Woodworth, C. D. & Sokolov, I. Atomic force microscopy detects differences in the surface brush of normal and cancerous cells. Nat. Nanotechnol. 4, 389–393 (2009).

    Article  CAS  Google Scholar 

  69. Martinez-Martin, D. et al. Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy. PLoS ONE 7, e30204 (2012).

    Article  CAS  Google Scholar 

  70. Roos, W. H. et al. Mechanics of bacteriophage maturation. Proc. Natl Acad. Sci. USA 109, 2342–2347 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  72. Janmey, P. A., Georges, P. C. & Hvidt, S. Basic rheology for biologists. Methods Cell Biol. 83, 3–27 (2007).

    CAS  Google Scholar 

  73. Nawaz, S. et al. Cell visco-elasticity measured with AFM and optical trapping at sub-micrometer deformations. PLoS ONE 7, e45297 (2012).

    Article  CAS  Google Scholar 

  74. Medalsy, I. D. & Müller, D. J. Nanomechanical properties of proteins and membranes depend on loading rate and electrostatic interactions. ACS Nano 7, 2642–2650 (2013).

    Article  CAS  Google Scholar 

  75. Herruzo, E. T., Perrino, A. P. & Garcia, R. Fast nanomechanical spectroscopy of soft matter. Nat. Commun. 5, 3126 (2014).

    Article  CAS  Google Scholar 

  76. Stewart, M. P. et al. Wedged AFM-cantilevers for parallel plate cell mechanics. Methods 60, 186–194 (2013).

    Article  CAS  Google Scholar 

  77. Fischer-Friedrich, E., Hyman, A. A., Julicher, F., Müller, D. J. & Helenius, J. Quantification of surface tension and internal pressure generated by single mitotic cells. Sci. Rep. 4, 6213 (2014).

    Article  CAS  Google Scholar 

  78. Fischer-Friedrich, E. et al. Rheology of the active cell cortex in mitosis. Biophys. J. 111, 589–600 (2016).

    Article  CAS  Google Scholar 

  79. Lee, G. U., Kidwell, D. A. & Colton, R. J. Sensing discrete streptavidin–biotin interactions with atomic force microscopy. Langmuir 10, 354–357 (1994).

    Article  CAS  Google Scholar 

  80. Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular forces and energies between ligands and receptors. Science 266, 257–259 (1994).

    Article  CAS  Google Scholar 

  81. Baumann, F., Heucke, S. F., Pippig, D. A. & Gaub, H. E. Tip localization of an atomic force microscope in transmission microscopy with nanoscale precision. Rev. Sci. Instrum. 86, 035109 (2015).

    Article  CAS  Google Scholar 

  82. Evans, E. A. & Calderwood, D. A. Forces and bond dynamics in cell adhesion. Science 316, 1148–1153 (2007).

    Article  CAS  Google Scholar 

  83. Dudko, O. K., Hummer, G. & Szabo, A. Theory, analysis, and interpretation of single-molecule force spectroscopy experiments. Proc. Natl Acad. Sci. USA 105, 15755–15760 (2008).

    Article  CAS  Google Scholar 

  84. Friddle, R. W., Noy, A. & De Yoreo, J. J. Interpreting the widespread nonlinear force spectra of intermolecular bonds. Proc. Natl Acad. Sci. USA 109, 13573–13578 (2012).

    Article  CAS  Google Scholar 

  85. Woodside, M. T. & Block, S. M. Reconstructing folding energy landscapes by single-molecule force spectroscopy. Ann. Rev. Biophys. 43, 19–39 (2014).

    Article  CAS  Google Scholar 

  86. Perez-Jimenez, R. et al. Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat. Struct. Mol. Biol. 18, 592–596 (2011).

    Article  CAS  Google Scholar 

  87. Oberhauser, A. F., Hansma, P. K., Carrion-Vazquez, M. & Fernandez, J. M. Stepwise unfolding of titin under force-clamp atomic force microscopy. Proc. Natl Acad. Sci. USA 98, 468–472 (2001).

    Article  CAS  Google Scholar 

  88. Stahl, S. W., Puchner, E. M. & Gaub, H. E. Photothermal cantilever actuation for fast single-molecule force spectroscopy. Rev. Sci. Instrum. 80, 073702 (2009).

    Article  CAS  Google Scholar 

  89. Krieg, M., Helenius, J., Heisenberg, C. P. & Müller, D. J. A bond for a lifetime: employing membrane nanotubes from living cells to determine receptor–ligand kinetics. Angew. Chem. Int. Ed. 47, 9775–9777 (2008).

    Article  CAS  Google Scholar 

  90. Alsteens, D. et al. Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape. Nat. Methods 12, 845–851 (2015).

    Article  CAS  Google Scholar 

  91. Wildling, L. et al. Probing binding pocket of serotonin transporter by single molecular force spectroscopy on living cells. J. Biol. Chem. 287, 105–113 (2012).

    Article  CAS  Google Scholar 

  92. Friedrichs, J., Helenius, J. & Müller, D. J. Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy. Nat. Protoc. 5, 1353–1361 (2010).

    Article  CAS  Google Scholar 

  93. Schoeler, C. et al. Mapping mechanical force propagation through biomolecular complexes. Nano Lett. 15, 7370–7376 (2015).

    Article  CAS  Google Scholar 

  94. Sieben, C. et al. Influenza virus binds its host cell using multiple dynamic interactions. Proc. Natl Acad. Sci. USA 109, 13626–13631 (2012).

    Article  CAS  Google Scholar 

  95. Alsteens, D. et al. Nanomechanical mapping of first binding steps of a virus to animal cells. Nat. Nanotechnol. 12, 177–183 (2017). This paper maps virus-binding events on animal cells and simultaneously extracts the steps and free energy landscape of viral ligands binding to cell surface receptors.

    Article  CAS  Google Scholar 

  96. King, G. M., Carter, A. R., Churnside, A. B., Eberle, L. S. & Perkins, T. T. Ultrastable atomic force microscopy: atomic-scale stability and registration in ambient conditions. Nano Lett. 9, 1451–1456 (2009).

    Article  CAS  Google Scholar 

  97. Bull, M. S., Sullan, R. M., Li, H. & Perkins, T. T. Improved single molecule force spectroscopy using micromachined cantilevers. ACS Nano 8, 4984–4995 (2014).

    Article  CAS  Google Scholar 

  98. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M. & Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997). This paper characterizes the mechanically induced unfolding and reversible refolding of single protein domains using AFM-based SMFS.

    Article  CAS  Google Scholar 

  99. Bippes, C. A. & Müller, D. J. High-resolution atomic force microscopy and spectroscopy of native membrane proteins. Rep. Prog. Phys. 74, 086601 (2011).

    Article  CAS  Google Scholar 

  100. Puchner, E. M. & Gaub, H. E. Force and function: probing proteins with AFM-based force spectroscopy. Curr. Opin. Struct. Biol. 19, 605–614 (2009).

    Article  CAS  Google Scholar 

  101. Žoldák, G. & Rief, M. Force as a single molecule probe of multidimensional protein energy landscapes. Curr. Opin. Struct. Biol. 23, 48–57 (2013).

    Article  CAS  Google Scholar 

  102. Kawamura, S., Colozo, A. T., Ge, L., Müller, D. J. & Park, P. S. Structural, energetic, and mechanical perturbations in rhodopsin mutant that causes congenital stationary night blindness. J. Biol. Chem. 287, 21826–21835 (2012).

    Article  CAS  Google Scholar 

  103. Mashaghi, A. et al. Reshaping of the conformational search of a protein by the chaperone trigger factor. Nature 500, 98–101 (2013).

    Article  CAS  Google Scholar 

  104. Nunes, J. M., Mayer-Hartl, M., Hartl, F. U. & Müller, D. J. Action of the Hsp70 chaperone system observed with single proteins. Nat. Commun. 6, 6307 (2015).

    Article  CAS  Google Scholar 

  105. Park, P. S. et al. Stabilizing effect of Zn2+ in native bovine rhodopsin. J. Biol. Chem. 282, 11377–11385 (2007).

    Article  CAS  Google Scholar 

  106. Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000). This paper describes the mechanically induced stepwise unfolding of membrane proteins using AFM-based SMFS.

    Article  CAS  Google Scholar 

  107. Damaghi, M., Koster, S., Bippes, C. A., Yildiz, O. & Müller, D. J. One β-hairpin follows the other: exploring refolding pathways and kinetics of the transmembrane β-barrel protein OmpG. Angew. Chem. Int. Ed. 50, 7422–7424 (2011).

    Article  CAS  Google Scholar 

  108. Kessler, M., Gottschalk, K. E., Janovjak, H., Müller, D. J. & Gaub, H. E. Bacteriorhodopsin folds into the membrane against an external force. J. Mol. Biol. 357, 644–654 (2006).

    Article  CAS  Google Scholar 

  109. Thoma, J., Bosshart, P., Pfreundschuh, M. & Müller, D. J. Out but not in: the large transmembrane β-barrel protein FhuA unfolds but cannot refold via β-hairpins. Structure 20, 2185–2190 (2012).

    Article  CAS  Google Scholar 

  110. Serdiuk, T. et al. YidC assists the stepwise and stochastic folding of membrane proteins. Nat. Chem. Biol. 12, 911–917 (2016).

    Article  CAS  Google Scholar 

  111. Thoma, J., Burmann, B. M., Hiller, S. & Müller, D. J. Impact of holdase chaperones Skp and SurA on the folding of beta-barrel outer-membrane proteins. Nat. Struct. Mol. Biol. 22, 795–802 (2015).

    Article  CAS  Google Scholar 

  112. Struckmeier, J. et al. Fully automated single-molecule force spectroscopy for screening applications. Nanotechnology 19, 384020 (2008).

    Article  CAS  Google Scholar 

  113. Otten, M. et al. From genes to protein mechanics on a chip. Nat. Methods 11, 1127–1130 (2014).

    Article  CAS  Google Scholar 

  114. Friedrichs, J. et al. A practical guide to quantify cell adhesion using single-cell force spectroscopy. Methods 60, 169–178 (2013).

    Article  CAS  Google Scholar 

  115. Benoit, M., Gabriel, D., Gerisch, G. & Gaub, H. E. Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nat. Cell Biol. 2, 313–317 (2000). This paper introduces AFM-based force spectroscopy to quantify the adhesive forces established by living cells.

    Article  CAS  Google Scholar 

  116. Ulrich, F. et al. Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin. Dev. Cell 9, 555–564 (2005).

    Article  CAS  Google Scholar 

  117. Te Riet, J. et al. Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6. J. Cell Sci. 127, 1595–1606 (2014).

    Article  CAS  Google Scholar 

  118. Alsteens, D., Van Dijck, P., Lipke, P. N. & Dufrêne, Y. F. Quantifying the forces driving cell–cell adhesion in a fungal pathogen. Langmuir 29, 13473–13480 (2013).

    Article  CAS  Google Scholar 

  119. Beaussart, A. et al. Single-cell force spectroscopy of probiotic bacteria. Biophys. J. 104, 1886–1892 (2013).

    Article  CAS  Google Scholar 

  120. Friedrichs, J., Helenius, J. & Müller, D. J. Stimulated single-cell force spectroscopy to quantify cell adhesion receptor crosstalk. Proteomics 10, 1455–1462 (2010).

    Article  CAS  Google Scholar 

  121. Chaudhuri, O., Parekh, S. H., Lam, W. A. & Fletcher, D. A. Combined atomic force microscopy and side-view optical imaging for mechanical studies of cells. Nat. Methods 6, 383–387 (2009).

    Article  CAS  Google Scholar 

  122. Gonnermann, C. et al. Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup. Integr. Biol. (Camb.) 7, 356–363 (2015).

    Article  CAS  Google Scholar 

  123. Ramanathan, S. P. et al. Cdk1-dependent mitotic enrichment of cortical myosin II promotes cell rounding against confinement. Nat. Cell Biol. 17, 148–159 (2015).

    Article  CAS  Google Scholar 

  124. Rouven Bruckner, B., Pietuch, A., Nehls, S., Rother, J. & Janshoff, A. Ezrin is a major regulator of membrane tension in epithelial cells. Sci. Rep. 5, 14700 (2015).

    Article  CAS  Google Scholar 

  125. Heu, C., Berquand, A., Elie-Caille, C. & Nicod, L. Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells. J. Struct. Biol. 178, 1–7 (2012).

    Article  CAS  Google Scholar 

  126. Hecht, F. M. et al. Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale. Soft Matter 11, 4584–4591 (2015).

    Article  CAS  Google Scholar 

  127. Formosa-Dague, C., Speziale, P., Foster, T. J., Geoghegan, J. A. & Dufrêne, Y. F. Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG. Proc. Natl Acad. Sci. USA 113, 410–415 (2016).

    Article  CAS  Google Scholar 

  128. Beaussart, A., El- Kirat-Chatel, S., Fontaine, T., Latge, J. P. & Dufrêne, Y. F. Nanoscale biophysical properties of the cell surface galactosaminogalactan from the fungal pathogen Aspergillus fumigatus. Nanoscale 7, 14996–15004 (2015).

    Article  CAS  Google Scholar 

  129. Dong, M., Husale, S. & Sahin, O. Determination of protein structural flexibility by microsecond force spectroscopy. Nat. Nanotechnol. 4, 514–517 (2009).

    Article  CAS  Google Scholar 

  130. Medalsy, I., Hensen, U. & Müller, D. J. Imaging and quantifying chemical and physical properties of native proteins at molecular resolution by force–volume AFM. Angew. Chem. Int. Ed. 50, 12103–12108 (2011).

    Article  CAS  Google Scholar 

  131. Wegmann, S., Medalsy, I. D., Mandelkow, E. & Müller, D. J. The fuzzy coat of pathological human Tau fibrils is a two-layered polyelectrolyte brush. Proc. Natl Acad. Sci. USA 110, E313–E321 (2013).

    Article  CAS  Google Scholar 

  132. Zhang, S. et al. Coexistence of ribbon and helical fibrils originating from hIAPP(20–29) revealed by quantitative nanomechanical atomic force microscopy. Proc. Natl Acad. Sci. USA 110, 2798–2803 (2013).

    Article  CAS  Google Scholar 

  133. Grandbois, M., Dettmann, W., Benoit, M. & Gaub, H. E. Affinity imaging of red blood cells using an atomic force microscope. J. Histochem. Cytochem. 48, 719–724 (2000).

    Article  CAS  Google Scholar 

  134. Dague, E. et al. Chemical force microscopy of single live cells. Nano Lett. 7, 3026–3030 (2007).

    Article  CAS  Google Scholar 

  135. Dupres, V. et al. The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo. Nat. Chem. Biol. 5, 857–862 (2009).

    Article  CAS  Google Scholar 

  136. Guo, S. F. et al. Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample. Nat. Nanotechnol. 11, 817–823 (2016).

    Article  CAS  Google Scholar 

  137. Pfreundschuh, M., Hensen, U. & Müller, D. J. Quantitative imaging of the electrostatic field and potential generated by a transmembrane protein pore at subnanometer resolution. Nano Lett. 13, 5585–5593 (2013).

    Article  CAS  Google Scholar 

  138. Alsteens, D., Trabelsi, H., Soumillion, P. & Dufrêne, Y. F. Multiparametric atomic force microscopy imaging of single bacteriophages extruding from living bacteria. Nat. Commun. 4, 2926 (2013).

    Article  CAS  Google Scholar 

  139. Pfreundschuh, M. et al. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM. Nat. Commun. 6, 8857 (2015).

    Article  CAS  Google Scholar 

  140. Pfreundschuh, M., Alsteens, D., Hilbert, M., Steinmetz, M. O. & Müller, D. J. Localizing chemical groups while imaging single native proteins by high-resolution atomic force microscopy. Nano Lett. 14, 2957–2964 (2014).

    Article  CAS  Google Scholar 

  141. Kim, D. & Sahin, O. Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy. Nat. Nanotechnol. 10, 264–269 (2015).

    Article  CAS  Google Scholar 

  142. Dong, M. & Sahin, O. A nanomechanical interface to rapid single-molecule interactions. Nat. Commun. 2, 247 (2011).

    Article  CAS  Google Scholar 

  143. Thomas, W. E., Vogel, V. & Sokurenko, E. Biophysics of catch bonds. Ann. Rev. Biophys. 37, 399–416 (2008).

    Article  CAS  Google Scholar 

  144. Janovjak, H., Struckmeier, J. & Müller, D. J. Hydrodynamic effects in fast AFM single-molecule force measurements. Eur. Biophys. J. 34, 91–96 (2005).

    Article  CAS  Google Scholar 

  145. Amo, C. A. & Garcia, R. Fundamental high-speed limits in single-molecule, single-cell, and nanoscale force spectroscopies. ACS Nano 10, 7117–7124 (2016).

    Article  CAS  Google Scholar 

  146. Rico, F., Gonzalez, L., Casuso, I., Puig-Vidal, M. & Scheuring, S. High-speed force spectroscopy unfolds titin at the velocity of molecular dynamics simulations. Science 342, 741–743 (2013).

    Article  CAS  Google Scholar 

  147. Fritz, J. et al. Translating biomolecular recognition into nanomechanics. Science 288, 316–318 (2000). This study introduces the use of microcantilevers to sense biomolecular binding.

    Article  CAS  Google Scholar 

  148. McKendry, R. et al. Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array. Proc. Natl Acad. Sci. USA 99, 9783–9788 (2002).

    Article  CAS  Google Scholar 

  149. Zhang, J. et al. Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA. Nat. Nanotechnol. 1, 214–220 (2006).

    Article  CAS  Google Scholar 

  150. Braun, T. et al. Quantitative time-resolved measurement of membrane protein–ligand interactions using microcantilever array sensors. Nat. Nanotechnol. 4, 179–185 (2009).

    Article  CAS  Google Scholar 

  151. Ndieyira, J. W. et al. Surface-stress sensors for rapid and ultrasensitive detection of active free drugs in human serum. Nat. Nanotechnol. 9, 225–232 (2014).

    Article  CAS  Google Scholar 

  152. Patil, S. B. et al. Decoupling competing surface binding kinetics and reconfiguration of receptor footprint for ultrasensitive stress assays. Nat. Nanotechnol. 10, 899–907 (2015).

    Article  CAS  Google Scholar 

  153. Huber, F., Lang, H. P., Backmann, N., Rimoldi, D. & Gerber, C. Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays. Nat. Nanotechnol. 8, 125–129 (2013).

    Article  CAS  Google Scholar 

  154. Huber, F. et al. Fast diagnostics of BRAF mutations in biopsies from malignant melanoma. Nano Lett. 16, 5373–5377 (2016).

    Article  CAS  Google Scholar 

  155. Barnes, J. R., Stephenson, R. J., Welland, M. E., Gerber, C. & Gimzewski, J. K. Photothermal spectroscopy with femtojoule sensitivity using a micromechanical device. Nature 372, 79–81 (1994).

    Article  CAS  Google Scholar 

  156. Kasas, S. et al. Detecting nanoscale vibrations as signature of life. Proc. Natl Acad. Sci. USA 112, 378–381 (2015).

    Article  CAS  Google Scholar 

  157. Carbonell, C. & Braunschweig, A. B. Toward 4D nanoprinting with tip-induced organic surface reactions. Acc. Chem. Res. http://dx.doi.org/10.1021/acs.accounts.6b00307 (2016).

  158. Tinazli, A., Piehler, J., Beuttler, M., Guckenberger, R. & Tampe, R. Native protein nanolithography that can write, read and erase. Nat. Nanotechnol. 2, 220–225 (2007).

    Article  CAS  Google Scholar 

  159. Martinez, R. V. et al. Large-scale nanopatterning of single proteins used as carriers of magnetic nanoparticles. Adv. Mater. 22, 588–591 (2010).

    Article  CAS  Google Scholar 

  160. Felts, J. R., Onses, M. S., Rogers, J. A. & King, W. P. Nanometer scale alignment of block-copolymer domains by means of a scanning probe tip. Adv. Mater. 26, 2999–3002 (2014).

    Article  CAS  Google Scholar 

  161. Shi, J., Chen, J. & Cremer, P. S. Sub-100 nm patterning of supported bilayers by nanoshaving lithography. J. Am. Chem. Soc. 130, 2718–2719 (2008).

    Article  CAS  Google Scholar 

  162. Cisneros, D. A., Friedrichs, J., Taubenberger, A., Franz, C. M. & Müller, D. J. Creating ultrathin nanoscopic collagen matrices for biological and biotechnological applications. Small 3, 956–963 (2007).

    Article  CAS  Google Scholar 

  163. Szoszkiewicz, R. et al. High-speed, sub-15 nm feature size thermochemical nanolithography. Nano Lett. 7, 1064–1069 (2007).

    Article  CAS  Google Scholar 

  164. Paul, P. C., Knoll, A. W., Holzner, F., Despont, M. & Duerig, U. Rapid turnaround scanning probe nanolithography. Nanotechnology 22, 275306 (2011).

    Article  CAS  Google Scholar 

  165. Gotsmann, B., Duerig, U., Frommer, J. & Hawker, C. J. Exploiting chemical switching in a Diels–Alder polymer for nanoscale probe lithography and data storage. Adv. Funct. Mater. 16, 1499–1505 (2006).

    Article  CAS  Google Scholar 

  166. Milner, A. A., Zhang, K. & Prior, Y. Floating tip nanolithography. Nano Lett. 8, 2017–2022 (2008).

    Article  CAS  Google Scholar 

  167. Carroll, K. M. et al. Fabricating nanoscale chemical gradients with ThermoChemical NanoLithography. Langmuir 29, 8675–8682 (2013).

    Article  CAS  Google Scholar 

  168. Jaschke, M. et al. The atomic force microscope as a tool to study and manipulate local surface properties. Biosens. Bioelectron. 11, 601–612 (1996).

    Article  CAS  Google Scholar 

  169. Salaita, K., Wang, Y. & Mirkin, C. A. Applications of dip-pen nanolithography. Nat. Nanotechnol. 2, 145–155 (2007).

    Article  CAS  Google Scholar 

  170. Lenhert, S., Mirkin, C. A. & Fuchs, H. In situ lipid dip-pen nanolithography under water. Scanning 32, 15–23 (2010).

    CAS  Google Scholar 

  171. Kim, K. H., Moldovan, N. & Espinosa, H. D. A nanofountain probe with sub-100 nm molecular writing resolution. Small 1, 632–635 (2005).

    Article  CAS  Google Scholar 

  172. Onses, M. S. et al. Hierarchical patterns of three-dimensional block-copolymer films formed by electrohydrodynamic jet printing and self-assembly. Nat. Nanotechnol. 8, 667–675 (2013).

    Article  CAS  Google Scholar 

  173. Lenhert, S. et al. Lipid multilayer gratings. Nat. Nanotechnol. 5, 275–279 (2010).

    Article  CAS  Google Scholar 

  174. Huo, F. et al. Polymer pen lithography. Science 321, 1658–1660 (2008).

    Article  CAS  Google Scholar 

  175. Albrecht, C. et al. DNA: a programmable force sensor. Science 301, 367–370 (2003).

    Article  CAS  Google Scholar 

  176. Pippig, D. A., Baumann, F., Strackharn, M., Aschenbrenner, D. & Gaub, H. E. Protein–DNA chimeras for nano assembly. ACS Nano 8, 6551–6555 (2014).

    Article  CAS  Google Scholar 

  177. Kufer, S. K. et al. Optically monitoring the mechanical assembly of single molecules. Nat. Nanotechnol. 4, 45–49 (2009).

    Article  CAS  Google Scholar 

  178. Puchner, E. M., Kufer, S. K., Strackharn, M., Stahl, S. W. & Gaub, H. E. Nanoparticle self-assembly on a DNA-scaffold written by single-molecule cut-and-paste. Nano Lett. 8, 3692–3695 (2008).

    Article  CAS  Google Scholar 

  179. Strackharn, M., Stahl, S. W., Puchner, E. M. & Gaub, H. E. Functional assembly of aptamer binding sites by single-molecule cut-and-paste. Nano Lett. 12, 2425–2428 (2012).

    Article  CAS  Google Scholar 

  180. Franz, C. M. & Müller, D. J. Analysing focal adhesion structure by AFM. J. Cell Sci. 118, 5315–5323 (2005).

    Article  CAS  Google Scholar 

  181. Cordes, T. et al. Resolving single-molecule assembled patterns with superresolution blink-microscopy. Nano Lett. 10, 645–651 (2010).

    Article  CAS  Google Scholar 

  182. Monserrate, A., Casado, S. & Flors, C. Correlative atomic force microscopy and localization-based super-resolution microscopy: revealing labelling and image reconstruction artefacts. Chemphyschem 15, 647–650 (2014).

    Article  CAS  Google Scholar 

  183. Fukuda, S. et al. High-speed atomic force microscope combined with single-molecule fluorescence microscope. Rev. Sci. Instrum. 84, 073706 (2013).

    Article  CAS  Google Scholar 

  184. Schmid, T., Opilik, L., Blum, C. & Zenobi, R. Nanoscale chemical imaging using tip-enhanced Raman spectroscopy: a critical review. Angew. Chem. Int. Ed. 52, 5940–5954 (2013).

    Article  CAS  Google Scholar 

  185. Berweger, S. et al. Nano-chemical infrared imaging of membrane proteins in lipid bilayers. J. Am. Chem. Soc. 135, 18292–18295 (2013).

    Article  CAS  Google Scholar 

  186. Ruggeri, F. S. et al. Infrared nanospectroscopy characterization of oligomeric and fibrillar aggregates during amyloid formation. Nat. Commun. 6, 7831 (2015).

    Article  CAS  Google Scholar 

  187. Hansma, P. K., Drake, B., Marti, O., Gould, S. A. & Prater, C. B. The scanning ion-conductance microscope. Science 243, 641–643 (1989). This classic study describes the invention of scanning ion conductance microscopy.

    Article  CAS  Google Scholar 

  188. Novak, P. et al. Nanoscale live-cell imaging using hopping probe ion conductance microscopy. Nat. Methods 6, 279–281 (2009).

    Article  CAS  Google Scholar 

  189. Novak, P. et al. Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels. Neuron 79, 1067–1077 (2013).

    Article  CAS  Google Scholar 

  190. Takahashi, Y. et al. Multifunctional nanoprobes for nanoscale chemical imaging and localized chemical delivery at surfaces and interfaces. Angew. Chem. Int. Ed. 50, 9638–9642 (2011).

    Article  CAS  Google Scholar 

  191. Shevchuk, A. I. et al. An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy. J. Cell Biol. 197, 499–508 (2012).

    Article  CAS  Google Scholar 

  192. Novak, P. et al. Imaging single nanoparticle interactions with human lung cells using fast ion conductance microscopy. Nano Lett. 14, 1202–1207 (2014).

    Article  CAS  Google Scholar 

  193. Shevchuk, A. I. et al. Imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy. Angew. Chem. Int. Ed. 45, 2212–2216 (2006).

    Article  CAS  Google Scholar 

  194. Klausen, L. H., Fuhs, T. & Dong, M. Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy. Nat. Commun. 7, 12447 (2016).

    Article  CAS  Google Scholar 

  195. Ossola, D. et al. Simultaneous scanning ion conductance microscopy and atomic force microscopy with microchanneled cantilevers. Phys. Rev. Lett. 115, 238103 (2015).

    Article  CAS  Google Scholar 

  196. Leo-Macias, A. et al. Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc. Nat. Commun. 7, 10342 (2016).

    Article  CAS  Google Scholar 

  197. Galvagnion, C. et al. Lipid vesicles trigger alpha-synuclein aggregation by stimulating primary nucleation. Nat. Chem. Biol. 11, 229–234 (2015).

    Article  CAS  Google Scholar 

  198. Lind, T. K., Zielinska, P., Wacklin, H. P., Urbanczyk-Lipkowska, Z. & Cardenas, M. Continuous flow atomic force microscopy imaging reveals fluidity and time-dependent interactions of antimicrobial dendrimer with model lipid membranes. ACS Nano 8, 396–408 (2014).

    Article  CAS  Google Scholar 

  199. Ko, S. H. et al. Synergistic self-assembly of RNA and DNA molecules. Nat. Chem. 2, 1050–1055 (2010).

    Article  CAS  Google Scholar 

  200. Sapra, K. T. et al. One beta hairpin after the other: exploring mechanical unfolding pathways of the transmembrane beta-barrel protein OmpG. Angew. Chem. Int. Ed. 48, 8306–8308 (2009).

    Article  CAS  Google Scholar 

  201. Strackharn, M., Pippig, D. A., Meyer, P., Stahl, S. W. & Gaub, H. E. Nanoscale arrangement of proteins by single-molecule cut-and-paste. J. Am. Chem. Soc. 134, 15193–15196 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

D.A. was supported by the Belgian National Foundation for Scientific Research (FNRS) and the Université catholique de Louvain (Fonds Spéciaux de Recherche). D.A. is a Research Associate FNRS. D.J.M. was supported by the Swiss National Science Foundation (SNF; grant 310030B_160225) and the NCCR Molecular Systems Engineering. C.G. and D.J.M. were supported by the Swiss Nanoscience Institute. H.E.G. acknowledges financial support by the ERC grant CelluFuel.

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Alsteens, D., Gaub, H., Newton, R. et al. Atomic force microscopy-based characterization and design of biointerfaces. Nat Rev Mater 2, 17008 (2017). https://doi.org/10.1038/natrevmats.2017.8

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