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Force spectroscopy of single cells using atomic force microscopy

Nature Reviews Methods Primers volume 1, Article number: 63 (2021) Cite this article

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Abstract

Physical forces and mechanical properties have critical roles in cellular function, physiology and disease. Over the past decade, atomic force microscopy (AFM) techniques have enabled substantial advances in our understanding of the tight relationship between force, mechanics and function in living cells and contributed to the growth of mechanobiology. In this Primer, we provide a comprehensive overview of the use of AFM-based force spectroscopy (AFM-FS) to study the strength and dynamics of cell adhesion from the cellular to the single-molecule level, spatially map cell surface receptors and quantify how cells dynamically regulate their mechanical and adhesive properties. We first introduce the importance of force and mechanics in cell biology and the general principles of AFM-FS methods. We describe procedures for sample and AFM probe preparations, the various AFM-FS modalities currently available and their respective advantages and limitations. We also provide details and recommendations for best usage practices, and discuss data analysis, statistics and reproducibility. We then exemplify the potential of AFM-FS in cellular and molecular biology with a series of recent successful applications focusing on viruses, bacteria, yeasts and mammalian cells. Finally, we speculate on the grand challenges in the area for the next decade.

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Fig. 1: General principles of AFM-FS.
Fig. 2: Schematic representation of various surface functionalization strategies to anchor biomolecules on inorganic surfaces for single-molecule force spectroscopy.
Fig. 3: Multiparametric AFM-FS and confocal imaging as a platform for probing the properties and interactions of living animal cells.
Fig. 4: Force–distance curve analysis.
Fig. 5: Single-molecule Dbp–ECM bond analysis reveals force-tuned dissociation paths.
Fig. 6: Ultra-strong binding forces of staphylococcal adhesins.
Fig. 7: Characterizing mammalian cell adhesion and mechanics using AFM-FS.
Fig. 8: Correlated AFM multiparametric and confocal imaging of single-virus binding interactions with living host cells.

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References

  1. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    Google Scholar 

  2. Schwartz, M. A. The force is with us. Science 323, 588–589 (2009).

    Google Scholar 

  3. Roca-Cusachs, P., Conte, V. & Trepat, X. Quantifying forces in cell biology. Nat. Cell Biol. 19, 742–751 (2017).

    Google Scholar 

  4. Petridou, N. I., Spiró, Z. & Heisenberg, C.-P. Multiscale force sensing in development. Nat. Cell Biol. 19, 581–588 (2017).

    Google Scholar 

  5. Huse, M. Mechanical forces in the immune system. Nat. Rev. Immunol. 17, 679–690 (2017).

    Google Scholar 

  6. Schwarz, U. S. Mechanobiology by the numbers: a close relationship between biology and physics. Nat. Rev. Mol. Cell Biol. 18, 711–712 (2017).

    Google Scholar 

  7. Ladoux, B. & Mège, R.-M. Mechanobiology of collective cell behaviours. Nat. Rev. Mol. Cell Biol. 18, 743–757 (2017).

    Google Scholar 

  8. Vining, K. H. & Mooney, D. J. Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 18, 728–742 (2017).

    Google Scholar 

  9. Dufrêne, Y. F. & Persat, A. Mechanomicrobiology: how bacteria sense and respond to forces. Nat. Rev. Microbiol. 18, 227–240 (2020).

    Google Scholar 

  10. Romani, P., Valcarcel-Jimenez, L., Frezza, C. & Dupont, S. Crosstalk between mechanotransduction and metabolism. Nat. Rev. Mol. Cell Biol. 22, 22–38 (2021).

    Google Scholar 

  11. Kechagia, J. Z., Ivaska, J. & Roca-Cusachs, P. Integrins as biomechanical sensors of the microenvironment. Nat. Rev. Mol. Cell Biol. 20, 457–473 (2019).

    Google Scholar 

  12. Uhler, C. & Shivashankar, G. V. Regulation of genome organization and gene expression by nuclear mechanotransduction. Nat. Rev. Mol. Cell Biol. 18, 717–727 (2017).

    Google Scholar 

  13. 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).

    Google Scholar 

  14. Finer, J. T., Simmons, R. M. & Spudich, J. A. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368, 113–119 (1994).

    ADS  Google Scholar 

  15. Schnitzer, M. J., Visscher, K. & Block, S. M. Force production by single kinesin motors. Nat. Cell Biol. 2, 718–723 (2000).

    Google Scholar 

  16. 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).

    Google Scholar 

  17. Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000).

    ADS  Google Scholar 

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

    Google Scholar 

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

    ADS  Google Scholar 

  20. Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H. E. How strong is a covalent bond? Science 283, 1727–1730 (1999).

    ADS  Google Scholar 

  21. Ashkin, A., Dziedzic, J. M. & Yamane, T. Optical trapping and manipulation of single cells using infrared laser beams. Nature 330, 769–771 (1987).

    ADS  Google Scholar 

  22. Neuman, K. C. & Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 5, 491–505 (2008).

    Google Scholar 

  23. Bian, K. et al. Scanning probe microscopy. Nat. Rev. Methods Prim. 1, 1–29 (2021).

    ADS  Google Scholar 

  24. Helenius, J., Heisenberg, C.-P., Gaub, H. E. & Muller, D. J. Single-cell force spectroscopy. J. Cell Sci. 121, 1785–1791 (2008).

    Google Scholar 

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

    ADS  Google Scholar 

  26. Puchner, E. M. et al. Mechanoenzymatics of titin kinase. Proc. Natl Acad. Sci. USA 105, 13385–13390 (2008).

    ADS  Google Scholar 

  27. del Rio, A. et al. Stretching single talin rod molecules activates vinculin binding. Science 323, 638–641 (2009).

    ADS  Google Scholar 

  28. Ivanovska, I. L. et al. Bacteriophage capsids: tough nanoshells with complex elastic properties. Proc. Natl Acad. Sci. USA 101, 7600–7605 (2004).

    ADS  Google Scholar 

  29. O’Callaghan, R., Job, K. M., Dull, R. O. & Hlady, V. Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy. Am. J. Physiol. Lung Cell Mol. Physiol. 301, L353–L360 (2011).

    Google Scholar 

  30. Howard, J., Grill, S. W. & Bois, J. S. Turing’s next steps: the mechanochemical basis of morphogenesis. Nat. Rev. Mol. Cell Biol. 12, 392–398 (2011).

    Google Scholar 

  31. Krieg, M. et al. Atomic force microscopy-based mechanobiology. Nat. Rev. Phys. 1, 41 (2019).

    Google Scholar 

  32. 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).

    Google Scholar 

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

    ADS  Google Scholar 

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

    ADS  Google Scholar 

  35. 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).

    ADS  Google Scholar 

  36. Wirtz, D., Konstantopoulos, K. & Searson, P. C. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nat. Rev. Cancer 11, 512–522 (2011).

    Google Scholar 

  37. Gaboriaud, F., Bailet, S., Dague, E. & Jorand, F. Surface structure and nanomechanical properties of Shewanella putrefaciens bacteria at two pH values (4 and 10) determined by atomic force microscopy. J. Bacteriol. 187, 3864–3868 (2005).

    Google Scholar 

  38. Touhami, A., Nysten, B. & Dufrêne, Y. F. Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy. Langmuir 19, 4539–4543 (2003).

    Google Scholar 

  39. Oh, Y. J. et al. Characterization of curli A production on living bacterial surfaces by scanning probe microscopy. Biophys. J. 103, 1666–1671 (2012).

    ADS  Google Scholar 

  40. Mandriota, N. et al. Cellular nanoscale stiffness patterns governed by intracellular forces. Nat. Mater. 18, 1071–1077 (2019).

    ADS  Google Scholar 

  41. Dufrêne, Y. F. et al. Imaging modes of atomic force microscopy for application in molecular and cell biology. Nat. Nanotechnol. 12, 295–307 (2017).

    ADS  Google Scholar 

  42. Butt, H. J. et al. Imaging cells with the atomic force microscope. J. Struct. Biol. 105, 54–61 (1990).

    Google Scholar 

  43. Hörber, J. K. et al. Investigation of living cells in the nanometer regime with the scanning force microscope. Scanning Microsc. 6, 919–929 (1992); discussion 929–930.

    Google Scholar 

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

    ADS  Google Scholar 

  45. 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).

    ADS  Google Scholar 

  46. Cappella, B. & Dietler, G. Force–distance curves by atomic force microscopy. Surf. Sci. Rep. 34, 1–104 (1999).

    ADS  Google Scholar 

  47. Radmacher, M., Cleveland, J. P., Fritz, M., Hansma, H. G. & Hansma, P. K. Mapping interaction forces with the atomic force microscope. Biophys. J. 66, 2159–2165 (1994).

    ADS  Google Scholar 

  48. Heinz, W. F. & Hoh, J. H. Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope. Trends Biotechnol. 17, 143–150 (1999).

    Google Scholar 

  49. Dufrêne, Y. F., Martínez-Martín, 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).

    Google Scholar 

  50. Adamcik, J., Berquand, A. & Mezzenga, R. Single-step direct measurement of amyloid fibrils stiffness by peak force quantitative nanomechanical atomic force microscopy. Appl. Phys. Lett. 98, 193701 (2011).

    ADS  Google Scholar 

  51. Rico, F., Su, C. & Scheuring, S. Mechanical mapping of single membrane proteins at submolecular resolution. Nano Lett. 11, 3983–3986 (2011).

    ADS  Google Scholar 

  52. Medalsy, I., Hensen, U. & Muller, 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).

    Google Scholar 

  53. 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).

    Google Scholar 

  54. Alsteens, D. et al. High-resolution imaging of chemical and biological sites on living cells using peak force tapping atomic force microscopy. Langmuir 28, 16738–16744 (2012).

    Google Scholar 

  55. Haupt, B. J., Pelling, A. E. & Horton, M. A. Integrated confocal and scanning probe microscopy for biomedical research. ScientificWorldJournal 6, 1609–1618 (2006).

    Google Scholar 

  56. Newton, R. et al. Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces. Nat. Protoc. 12, 2275–2292 (2017).

    Google Scholar 

  57. Cumpson, P. J. & Hedley, J. Accurate analytical measurements in the atomic force microscope: a microfabricated spring constant standard potentially traceable to the SI. Nanotechnology 14, 1279–1288 (2003).

    ADS  Google Scholar 

  58. Sader, J. E., Chon, J. W. M. & Mulvaney, P. Calibration of rectangular atomic force microscope cantilevers. Rev. Sci. Instrum. 70, 3967–3969 (1999).

    ADS  Google Scholar 

  59. Hutter, J. L. & Bechhoefer, J. Calibration of atomic‐force microscope tips. Rev. Sci. Instrum. 64, 1868–1873 (1993).

    ADS  Google Scholar 

  60. Shoelson, B., Dimitriadis, E. K., Cai, H., Kachar, B. & Chadwick, R. S. Evidence and implications of inhomogeneity in tectorial membrane elasticity. Biophys. J. 87, 2768–2777 (2004).

    ADS  Google Scholar 

  61. Lin, D. C., Dimitriadis, E. K. & Horkay, F. Robust strategies for automated AFM force curve analysis — I. Non-adhesive indentation of soft, inhomogeneous materials. J. Biomech. Eng. 129, 430–440 (2006).

    Google Scholar 

  62. Lin, D. C., Dimitriadis, E. K. & Horkay, F. Robust strategies for automated AFM force curve analysis — II: adhesion-influenced indentation of soft, elastic materials. J. Biomech. Eng. 129, 904–912 (2007).

    Google Scholar 

  63. Rudoy, D., Yuen, S. G., Howe, R. D. & Wolfe, P. J. Bayesian change-point analysis for atomic force microscopy and soft material indentation. J. R. Stat. Society. C. 59, 573–593 (2010).

    MathSciNet  Google Scholar 

  64. Benítez, R., Moreno-Flores, S., Bolós, V. J. & Toca-Herrera, J. L. A new automatic contact point detection algorithm for AFM force curves. Microsc. Res. Tech. 76, 870–876 (2013).

    Google Scholar 

  65. Gavara, N. Combined strategies for optimal detection of the contact point in AFM force–indentation curves obtained on thin samples and adherent cells. Sci. Rep. 6, 21267 (2016).

    ADS  Google Scholar 

  66. El Kirat, K., Burton, I., Dupres, V. & Dufrene, Y. F. Sample preparation procedures for biological atomic force microscopy. J. Microsc. 218, 199–207 (2005).

    MathSciNet  Google Scholar 

  67. Stewart, M. P., Toyoda, Y., Hyman, A. A. & Müller, D. J. Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp. Nat. Protoc. 7, 143–154 (2012).

    Google Scholar 

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

    Google Scholar 

  69. Efremov, Y. M., Cartagena-Rivera, A. X., Athamneh, A. I. M., Suter, D. M. & Raman, A. Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy. Nat. Protoc. 13, 2200–2216 (2018).

    Google Scholar 

  70. Formosa, C. et al. Generation of living cell arrays for atomic force microscopy studies. Nat. Protoc. 10, 199–204 (2015).

    Google Scholar 

  71. Beaussart, A. et al. Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy. Nat. Protoc. 9, 1049–1055 (2014).

    Google Scholar 

  72. Dufrêne, Y. F. Atomic force microscopy and chemical force microscopy of microbial cells. Nat. Protoc. 3, 1132–1138 (2008).

    Google Scholar 

  73. Berquand, A. et al. Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy. Langmuir 21, 5517–5523 (2005).

    Google Scholar 

  74. Kasas, S. & Ikai, A. A method for anchoring round shaped cells for atomic force microscope imaging. Biophys. J. 68, 1678–1680 (1995).

    ADS  Google Scholar 

  75. Dufrêne, Y. F., Boonaert, C. J., Gerin, P. A., Asther, M. & Rouxhet, P. G. Direct probing of the surface ultrastructure and molecular interactions of dormant and germinating spores of Phanerochaete chrysosporium. J. Bacteriol. 181, 5350–5354 (1999).

    Google Scholar 

  76. Bizzarri, A. R. & Cannistraro, S. The application of atomic force spectroscopy to the study of biological complexes undergoing a biorecognition process. Chem. Soc. Rev. 39, 734–749 (2010).

    Google Scholar 

  77. Ott, W., Jobst, M. A., Schoeler, C., Gaub, H. E. & Nash, M. A. Single-molecule force spectroscopy on polyproteins and receptor–ligand complexes: the current toolbox. J. Struct. Biol. 197, 3–12 (2017).

    Google Scholar 

  78. Müller, D. J. et al. Atomic force microscopy-based force spectroscopy and multiparametric imaging of biomolecular and cellular systems. Chem. Rev. https://doi.org/10.1021/acs.chemrev.0c00617 (2020).

    Article  Google Scholar 

  79. Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K. & Schindler, H. Detection and localization of individual antibody–antigen recognition events by atomic force microscopy. Proc. Natl Acad. Sci. USA 93, 3477–3481 (1996).

    ADS  Google Scholar 

  80. Kienberger, F., Ebner, A., Gruber, H. J. & Hinterdorfer, P. Molecular recognition imaging and force spectroscopy of single biomolecules. Acc. Chem. Res. 39, 29–36 (2006).

    Google Scholar 

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

    Google Scholar 

  82. Theile, C. S. et al. Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8, 1800–1807 (2013).

    Google Scholar 

  83. Chen, G., Ning, X., Park, B., Boons, G.-J. & Xu, B. Simple, clickable protocol for atomic force microscopy tip modification and its application for trace ricin detection by recognition imaging. Langmuir 25, 2860–2864 (2009).

    Google Scholar 

  84. Alsteens, D., Dague, E., Rouxhet, P. G., Baulard, A. R. & Dufrêne, Y. F. Direct measurement of hydrophobic forces on cell surfaces using AFM. Langmuir 23, 11977–11979 (2007).

    Google Scholar 

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

    ADS  Google Scholar 

  86. Viljoen, A., Viela, F., Kremer, L. & Dufrêne, Y. F. Fast chemical force microscopy demonstrates that glycopeptidolipids define nanodomains of varying hydrophobicity on mycobacteria. Nanoscale Horiz. 5, 944–953 (2020).

    ADS  Google Scholar 

  87. Dorobantu, L. S., Bhattacharjee, S., Foght, J. M. & Gray, M. R. Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity. Langmuir 24, 4944–4951 (2008).

    Google Scholar 

  88. 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).

    Google Scholar 

  89. Ng, G. et al. Receptor-independent, direct membrane binding leads to cell-surface lipid sorting and Syk kinase activation in dendritic cells. Immunity 29, 807–818 (2008).

    Google Scholar 

  90. Taubenberger, A. V., Hutmacher, D. W. & Muller, D. J. Single-cell force spectroscopy, an emerging tool to quantify cell adhesion to biomaterials. Tissue Eng. Part. B Rev. 20, 40–55 (2014).

    Google Scholar 

  91. Schubert, R. et al. Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy. FEBS Lett. 588, 3639–3648 (2014).

    Google Scholar 

  92. Le, D. T. L., Guérardel, Y., Loubière, P., Mercier-Bonin, M. & Dague, E. Measuring kinetic dissociation/association constants between Lactococcus lactis bacteria and mucins using living cell probes. Biophys. J. 101, 2843–2853 (2011).

    ADS  Google Scholar 

  93. Ovchinnikova, E. S., Krom, B. P., van der Mei, H. C. & Busscher, H. J. Force microscopic and thermodynamic analysis of the adhesion between Pseudomonas aeruginosa and Candida albicans. Soft Matter 8, 6454–6461 (2012).

    ADS  Google Scholar 

  94. Emerson, R. J. et al. Microscale correlation between surface chemistry, texture, and the adhesive strength of Staphylococcus epidermidis. Langmuir 22, 11311–11321 (2006).

    Google Scholar 

  95. Bowen, W. R., Lovitt, R. W. & Wright, C. J. Atomic force microscopy study of the adhesion of Saccharomyces cerevisiae. J. Colloid Interface Sci. 237, 54–61 (2001).

    ADS  Google Scholar 

  96. Razatos, A., Ong, Y. L., Sharma, M. M. & Georgiou, G. Molecular determinants of bacterial adhesion monitored by atomic force microscopy. Proc. Natl Acad. Sci. USA 95, 11059–11064 (1998).

    ADS  Google Scholar 

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

    ADS  Google Scholar 

  98. Guillaume-Gentil, O. et al. Force-controlled manipulation of single cells: from AFM to FluidFM. Trends Biotechnol. 32, 381–388 (2014).

    Google Scholar 

  99. Potthoff, E. et al. Rapid and serial quantification of adhesion forces of yeast and mammalian cells. PLoS ONE 7, e52712 (2012).

    ADS  Google Scholar 

  100. Potthoff, E., Ossola, D., Zambelli, T. & Vorholt, J. A. Bacterial adhesion force quantification by fluidic force microscopy. Nanoscale 7, 4070–4079 (2015).

    ADS  Google Scholar 

  101. Dehullu, J. et al. Fluidic force microscopy demonstrates that homophilic adhesion by Candida albicans Als proteins is mediated by amyloid bonds between cells. Nano Lett. 19, 3846–3853 (2019).

    ADS  Google Scholar 

  102. Mathelié-Guinlet, M. et al. Single-cell fluidic force microscopy reveals stress-dependent molecular interactions in yeast mating. Commun. Biol. 4, 1–8 (2021).

    Google Scholar 

  103. Dupres, V. et al. Nanoscale mapping and functional analysis of individual adhesins on living bacteria. Nat. Methods 2, 515–520 (2005).

    Google Scholar 

  104. Dumitru, A. C. et al. Label-free imaging of cholesterol assemblies reveals hidden nanomechanics of breast cancer cells. Adv. Sci. 7, 2002643 (2020).

    Google Scholar 

  105. Sokolov, I. & Dokukin, M. E. Imaging of soft and biological samples using AFM ringing mode. Methods Mol. Biol. 1814, 469–482 (2018).

    Google Scholar 

  106. Dokukin, M. E. & Sokolov, I. Nanoscale compositional mapping of cells, tissues, and polymers with ringing mode of atomic force microscopy. Sci. Rep. 7, 11828 (2017).

    ADS  Google Scholar 

  107. Stroh, C. M. et al. Simultaneous topography and recognition imaging using force microscopy. Biophys. J. 87, 1981–1990 (2004).

    ADS  Google Scholar 

  108. Stroh, C. et al. Single-molecule recognition imaging microscopy. Proc. Natl Acad. Sci. USA 101, 12503–12507 (2004).

    ADS  Google Scholar 

  109. Chtcheglova, L. A., Waschke, J., Wildling, L., Drenckhahn, D. & Hinterdorfer, P. Nano-scale dynamic recognition imaging on vascular endothelial cells. Biophys. J. 93, L11–L13 (2007).

    Google Scholar 

  110. Koehler, M. et al. Combined recognition imaging and force spectroscopy: a new mode for mapping and studying interaction sites at low lateral density. Sci. Adv. Mater. 9, 128–134 (2017).

    Google Scholar 

  111. Sahin, O., Magonov, S., Su, C., Quate, C. F. & Solgaard, O. An atomic force microscope tip designed to measure time-varying nanomechanical forces. Nat. Nanotechnol. 2, 507–514 (2007).

    ADS  Google Scholar 

  112. Cronin-Golomb, M. & Sahin, O. High-resolution nanomechanical analysis of suspended electrospun silk fibers with the torsional harmonic atomic force microscope. Beilstein J. Nanotechnol. 4, 243–248 (2013).

    Google Scholar 

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

    ADS  Google Scholar 

  114. Nievergelt, A. P., Banterle, N., Andany, S. H., Gönczy, P. & Fantner, G. E. High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6. Nat. Nanotechnol. 13, 696–701 (2018).

    ADS  Google Scholar 

  115. Nievergelt, A. P., Brillard, C., Eskandarian, H. A., McKinney, J. D. & Fantner, G. E. Photothermal off-resonance tapping for rapid and gentle atomic force imaging of live cells. Int. J. Mol. Sci. 19, E2984 (2018).

    Google Scholar 

  116. Dokukin, M. & Sokolov, I. High-resolution high-speed dynamic mechanical spectroscopy of cells and other soft materials with the help of atomic force microscopy. Sci. Rep. 5, 12630 (2015).

    ADS  Google Scholar 

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

    ADS  Google Scholar 

  118. Lozano, J. R. & Garcia, R. Theory of multifrequency atomic force microscopy. Phys. Rev. Lett. 100, 076102 (2008).

    ADS  Google Scholar 

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

    ADS  Google Scholar 

  120. Shekhawat, G. S. & Dravid, V. P. Nanoscale imaging of buried structures via scanning near-field ultrasound holography. Science 310, 89–92 (2005).

    ADS  Google Scholar 

  121. Tetard, L., Passian, A. & Thundat, T. New modes for subsurface atomic force microscopy through nanomechanical coupling. Nat. Nanotech 5, 105–109 (2010).

    ADS  Google Scholar 

  122. Liu, B., Chen, W., Evavold, B. D. & Zhu, C. Accumulation of dynamic catch bonds between TCR and agonist peptide–MHC triggers T cell signaling. Cell 157, 357–368 (2014).

    Google Scholar 

  123. 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).

    ADS  Google Scholar 

  124. Brujic, J., Hermans, R. I. Z., Garcia-Manyes, S., Walther, K. A. & Fernandez, J. M. Dwell-time distribution analysis of polyprotein unfolding using force-clamp spectroscopy. Biophys. J. 92, 2896–2903 (2007).

    ADS  Google Scholar 

  125. Garcia-Manyes, S., Kuo, T.-L. & Fernández, J. M. Contrasting the individual reactive pathways in protein unfolding and disulfide bond reduction observed within a single protein. J. Am. Chem. Soc. 133, 3104–3113 (2011).

    Google Scholar 

  126. Marshall, B. T. et al. Direct observation of catch bonds involving cell-adhesion molecules. Nature 423, 190–193 (2003).

    ADS  Google Scholar 

  127. Mathelié-Guinlet, M. et al. Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen. Nat. Commun. 11, 5431 (2020).

    ADS  Google Scholar 

  128. Mignolet, J., Mathelié-Guinlet, M., Viljoen, A. & Dufrêne, Y. F. AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction. Nanoscale Horiz. 6, 489–496 (2021).

    ADS  Google Scholar 

  129. Lecuit, T. & Lenne, P.-F. Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesis. Nat. Rev. Mol. Cell Biol. 8, 633–644 (2007).

    Google Scholar 

  130. Oberhauser, A. F., Marszalek, P. E., Erickson, H. P. & Fernandez, J. M. The molecular elasticity of the extracellular matrix protein tenascin. Nature 393, 181–185 (1998).

    ADS  Google Scholar 

  131. Fletcher, D. A. & Mullins, R. D. Cell mechanics and the cytoskeleton. Nature 463, 485–492 (2010).

    ADS  Google Scholar 

  132. 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).

    Google Scholar 

  133. Kollmannsberger, P. & Fabry, B. Linear and nonlinear rheology of living cells. Annu. Rev. Mater. Res. 41, 75–97 (2011).

    ADS  Google Scholar 

  134. Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B. & Chadwick, R. S. Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophysical J. 82, 2798–2810 (2002).

    ADS  Google Scholar 

  135. 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).

    Google Scholar 

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

    ADS  Google Scholar 

  137. Lomakin, A. J. et al. The nucleus acts as a ruler tailoring cell responses to spatial constraints. Science 370, eaba2894 (2020).

    Google Scholar 

  138. Fläschner, G., Roman, C. I., Strohmeyer, N., Martinez-Martin, D. & Müller, D. J. Rheology of rounded mammalian cells over continuous high-frequencies. Nat. Commun. 12, 2922 (2021).

    ADS  Google Scholar 

  139. Haase, K. & Pelling, A. E. Investigating cell mechanics with atomic force microscopy. J. R. Soc. Interface 12, 20140970 (2015).

    Google Scholar 

  140. 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).

    ADS  Google Scholar 

  141. Odermatt, P. D. et al. Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division. Nat. Phys. 16, 57–62 (2020).

    Google Scholar 

  142. Baumgartner, W., Hinterdorfer, P. & Schindler, H. Data analysis of interaction forces measured with the atomic force microscope. Ultramicroscopy 82, 85–95 (2000).

    Google Scholar 

  143. Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72, 1541–1555 (1997).

    Google Scholar 

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

    ADS  Google Scholar 

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

    Google Scholar 

  146. Sokurenko, E. V., Vogel, V. & Thomas, W. E. Catch-bond mechanism of force-enhanced adhesion: counterintuitive, elusive, but … widespread? Cell Host Microbe 4, 314–323 (2008).

    Google Scholar 

  147. Pietuch, A. & Janshoff, A. Mechanics of spreading cells probed by atomic force microscopy. Open. Biol. 3, 130084 (2013).

    Google Scholar 

  148. Dao, L., Gonnermann, C. & Franz, C. M. Investigating differential cell–matrix adhesion by directly comparative single-cell force spectroscopy. J. Mol. Recognit. 26, 578–589 (2013).

    Google Scholar 

  149. Strohmeyer, N., Bharadwaj, M., Costell, M., Fässler, R. & Müller, D. J. Fibronectin-bound α5β1 integrins sense load and signal to reinforce adhesion in less than a second. Nat. Mater. 16, 1262–1270 (2017).

    ADS  Google Scholar 

  150. Cui, Y. et al. Cyclic stretching of soft substrates induces spreading and growth. Nat. Commun. 6, 6333 (2015).

    ADS  Google Scholar 

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

    Google Scholar 

  152. Radmacher, M. Measuring the elastic properties of living cells by the atomic force microscope. Methods Cell Biol. 68, 67–90 (2002).

    Google Scholar 

  153. Krieg, M., Helenius, J., Heisenberg, C.-P. & Muller, 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).

    Google Scholar 

  154. Zhu, R. et al. Nanopharmacological force sensing to reveal allosteric coupling in transporter binding sites. Angew. Chem. Int. Ed. 55, 1719–1722 (2016).

    Google Scholar 

  155. Dufrêne, Y. F. Sticky microbes: forces in microbial cell adhesion. Trends Microbiol. 23, 376–382 (2015).

    Google Scholar 

  156. Janshoff, A., Neitzert, M., Oberdörfer, Y. & Fuchs, H. Force spectroscopy of molecular systems — single molecule spectroscopy of polymers and biomolecules. Angew. Chem. Int. Ed. 39, 3212–3237 (2000).

    ADS  Google Scholar 

  157. Odijk, T. Stiff chains and filaments under tension. Macromolecules 28, 7016–7018 (1995).

    ADS  Google Scholar 

  158. Rief, M., Oesterhelt, F., Heymann, B. & Gaub, H. E. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science 275, 1295–1297 (1997).

    Google Scholar 

  159. Marszalek, P. E., Oberhauser, A. F., Pang, Y. P. & Fernandez, J. M. Polysaccharide elasticity governed by chair–boat transitions of the glucopyranose ring. Nature 396, 661–664 (1998).

    ADS  Google Scholar 

  160. Lee, G. et al. Nanospring behaviour of ankyrin repeats. Nature 440, 246–249 (2006).

    ADS  Google Scholar 

  161. Beaussart, A. et al. Nanoscale adhesion forces of Pseudomonas aeruginosa type IV pili. ACS Nano 8, 10723–10733 (2014).

    Google Scholar 

  162. Kramers, H. A. Brownian motion in a field of force and the diffusion model of chemical reactions. Physica 7, 284–304 (1940).

    ADS  MathSciNet  MATH  Google Scholar 

  163. Sedlak, S. M., Schendel, L. C., Gaub, H. E. & Bernardi, R. C. Streptavidin/biotin: tethering geometry defines unbinding mechanics. Sci. Adv. 6, eaay5999 (2020).

    ADS  Google Scholar 

  164. Hummer, G. & Szabo, A. Free energy surfaces from single-molecule force spectroscopy. Acc. Chem. Res. 38, 504–513 (2005).

    Google Scholar 

  165. Hummer, G. & Szabo, A. Kinetics from nonequilibrium single-molecule pulling experiments. Biophys. J. 85, 5–15 (2003).

    ADS  Google Scholar 

  166. Dudko, O. K., Hummer, G. & Szabo, A. Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys. Rev. Lett. 96, 108101 (2006).

    ADS  Google Scholar 

  167. 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).

    ADS  Google Scholar 

  168. Battle, C. et al. Broken detailed balance at mesoscopic scales in active biological systems. Science 352, 604–607 (2016).

    ADS  Google Scholar 

  169. Dammer, U. et al. Binding strength between cell adhesion proteoglycans measured by atomic force microscopy. Science 267, 1173–1175 (1995).

    ADS  Google Scholar 

  170. Fritz, J., Katopodis, A. G., Kolbinger, F. & Anselmetti, D. Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy. Proc. Natl Acad. Sci. USA 95, 12283–12288 (1998).

    ADS  Google Scholar 

  171. Li, F., Redick, S. D., Erickson, H. P. & Moy, V. T. Force measurements of the α5β1 integrin–fibronectin interaction. Biophys. J. 84, 1252–1262 (2003).

    ADS  Google Scholar 

  172. Oh, Y. J. et al. Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds. Sci. Rep. 6, 33909 (2016).

    ADS  Google Scholar 

  173. Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy. Nature 397, 50–53 (1999).

    ADS  Google Scholar 

  174. Alsteens, D. et al. Unfolding individual Als5p adhesion proteins on live cells. ACS Nano 3, 1677–1682 (2009).

    Google Scholar 

  175. Alsteens, D., Garcia, M. C., Lipke, P. N. & Dufrêne, Y. F. Force-induced formation and propagation of adhesion nanodomains in living fungal cells. Proc. Natl Acad. Sci. USA 107, 20744–20749 (2010).

    ADS  Google Scholar 

  176. Strnad, M. et al. Nanomechanical mechanisms of Lyme disease spirochete motility enhancement in extracellular matrix. Commun. Biol. 4, 268 (2021).

    Google Scholar 

  177. Chilkoti, A., Boland, T., Ratner, B. D. & Stayton, P. S. The relationship between ligand-binding thermodynamics and protein–ligand interaction forces measured by atomic force microscopy. Biophys. J. 69, 2125–2130 (1995).

    ADS  Google Scholar 

  178. Herman-Bausier, P. & Dufrêne, Y. F. Force matters in hospital-acquired infections. Science 359, 1464–1465 (2018).

    ADS  Google Scholar 

  179. Viljoen, A., Mignolet, J., Viela, F., Mathelié-Guinlet, M. & Dufrêne, Y. F. How microbes use force to control adhesion. J. Bacteriol. 202, e00125-20 (2020).

    Google Scholar 

  180. Milles, L. F. & Gaub, H. E. Extreme mechanical stability in protein complexes. Curr. Opin. Struct. Biol. 60, 124–130 (2020).

    Google Scholar 

  181. Bernardi, R. C. et al. Mechanisms of nanonewton mechanostability in a protein complex revealed by molecular dynamics simulations and single-molecule force spectroscopy. J. Am. Chem. Soc. 141, 14752–14763 (2019).

    Google Scholar 

  182. Liu, Z. et al. High force catch bond mechanism of bacterial adhesion in the human gut. Nat. Commun. 11, 4321 (2020).

    ADS  Google Scholar 

  183. O’Connell, D. Dock, lock and latch. Nat. Rev. Microbiol. 1, 171–171 (2003).

    Google Scholar 

  184. Foster, T. J. Immune evasion by staphylococci. Nat. Rev. Microbiol. 3, 948–958 (2005).

    Google Scholar 

  185. Herman, P. et al. The binding force of the staphylococcal adhesin SdrG is remarkably strong. Mol. Microbiol. 93, 356–368 (2014).

    Google Scholar 

  186. Milles, L. F., Schulten, K., Gaub, H. E. & Bernardi, R. C. Molecular mechanism of extreme mechanostability in a pathogen adhesin. Science 359, 1527–1533 (2018).

    ADS  Google Scholar 

  187. Thomas, W. Catch bonds in adhesion. Annu. Rev. Biomed. Eng. 10, 39–57 (2008).

    Google Scholar 

  188. Herman-Bausier, P. et al. Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion. Proc. Natl Acad. Sci. USA 115, 5564–5569 (2018).

    Google Scholar 

  189. Viela, F., Speziale, P., Pietrocola, G. & Dufrêne, Y. F. Mechanostability of the fibrinogen bridge between staphylococcal surface protein ClfA and endothelial cell integrin αVβ3. Nano Lett. 19, 7400–7410 (2019).

    ADS  Google Scholar 

  190. Vitry, P. et al. Force-induced strengthening of the interaction between Staphylococcus aureus clumping factor B and Loricrin. mBio 8, e01748-17 (2017).

    Google Scholar 

  191. Yakovenko, O. et al. FimH forms catch bonds that are enhanced by mechanical force due to allosteric regulation. J. Biol. Chem. 283, 11596–11605 (2008).

    Google Scholar 

  192. Sauer, M. M. et al. Catch-bond mechanism of the bacterial adhesin FimH. Nat. Commun. 7, 10738 (2016).

    ADS  Google Scholar 

  193. Geoghegan, J. A., Foster, T. J., Speziale, P. & Dufrêne, Y. F. Live-cell nanoscopy in antiadhesion therapy. Trends Microbiol. 25, 512–514 (2017).

    Google Scholar 

  194. Feuillie, C. et al. Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC. Proc. Natl Acad. Sci. USA 114, 3738–3743 (2017).

    Google Scholar 

  195. Krieg, M. et al. Tensile forces govern germ-layer organization in zebrafish. Nat. Cell Biol. 10, 429–436 (2008).

    Google Scholar 

  196. Benito-Jardón, M. et al. αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation. J. Cell Biol. 219, e202004198 (2020).

    Google Scholar 

  197. Bharadwaj, M. et al. αV-Class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin. Nat. Commun. 8, 14348 (2017).

    ADS  Google Scholar 

  198. Spoerri, P. M., Strohmeyer, N., Sun, Z., Fässler, R. & Müller, D. J. Protease-activated receptor signalling initiates α5β1-integrin-mediated adhesion in non-haematopoietic cells. Nat. Mater. 19, 218–226 (2020).

    ADS  Google Scholar 

  199. Thompson, A. J. et al. Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain. eLife 8, e39356 (2019).

    Google Scholar 

  200. Koser, D. E. et al. Mechanosensing is critical for axon growth in the developing brain. Nat. Neurosci. 19, 1592–1598 (2016).

    Google Scholar 

  201. Moeendarbary, E. et al. The soft mechanical signature of glial scars in the central nervous system. Nat. Commun. 8, 14787 (2017).

    ADS  Google Scholar 

  202. Lopez, J. I., Kang, I., You, W.-K., McDonald, D. M. & Weaver, V. M. In situ force mapping of mammary gland transformation. Integr. Biol. 3, 910–921 (2011).

    Google Scholar 

  203. Acerbi, I. et al. Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration. Integr. Biol. 7, 1120–1134 (2015).

    Google Scholar 

  204. Plodinec, M. et al. The nanomechanical signature of breast cancer. Nat. Nanotechnol. 7, 757–765 (2012).

    ADS  Google Scholar 

  205. Xu, W. et al. Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells. PLoS ONE 7, e46609 (2012).

    ADS  Google Scholar 

  206. Yang, B. et al. Stopping transformed cancer cell growth by rigidity sensing. Nat. Mater. 19, 239–250 (2020).

    ADS  Google Scholar 

  207. Maller, O. et al. Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression. Nat. Mater. 20, 548–559 (2021).

    ADS  Google Scholar 

  208. Mekhdjian, A. H. et al. Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix. Mol. Biol. Cell 28, 1467–1488 (2017).

    Google Scholar 

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

    Google Scholar 

  210. Carneiro, F. A. et al. Probing the interaction between vesicular stomatitis virus and phosphatidylserine. Eur. Biophys. J. 35, 145–154 (2006).

    Google Scholar 

  211. Rankl, C. et al. Multiple receptors involved in human rhinovirus attachment to live cells. Proc. Natl Acad. Sci. U S A 105, 17778–17783 (2008).

    ADS  Google Scholar 

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

    Google Scholar 

  213. Alsteens, D. et al. Nanomechanical mapping of first binding steps of a virus to animal cells. Nat. Nanotechnol. 12, 177–183 (2017).

    ADS  Google Scholar 

  214. Delguste, M. et al. Single-virus force spectroscopy discriminates the intrinsic role of two viral glycoproteins upon cell surface attachment. Nano Lett. 21, 847–853 (2021).

    ADS  Google Scholar 

  215. Koehler, M. et al. Glycan-mediated enhancement of reovirus receptor binding. Nat. Commun. 10, 4460 (2019).

    ADS  Google Scholar 

  216. Koehler, M. et al. Reovirus directly engages integrin to recruit clathrin for entry into host cells. Nat. Commun. 12, 2149 (2021).

    ADS  Google Scholar 

  217. Oropesa-Nuñez, R., Mescola, A., Vassalli, M. & Canale, C. Impact of experimental parameters on cell–cell force spectroscopy signature. Sensors 21, 1069 (2021).

    ADS  Google Scholar 

  218. te Riet, J. et al. Interlaboratory round robin on cantilever calibration for AFM force spectroscopy. Ultramicroscopy 111, 1659–1669 (2011).

    Google Scholar 

  219. Schillers, H. et al. Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples. Sci. Rep. 7, 5117 (2017).

    ADS  Google Scholar 

  220. Sader, J. E. & White, L. Theoretical analysis of the static deflection of plates for atomic force microscope applications. J. Appl. Phys. 74, 1–9 (1993).

    ADS  Google Scholar 

  221. Yu, M., Strohmeyer, N., Wang, J., Müller, D. J. & Helenius, J. Increasing throughput of AFM-based single cell adhesion measurements through multisubstrate surfaces. Beilstein J. Nanotechnol. 6, 157–166 (2015).

    Google Scholar 

  222. Rheinlaender, J. et al. Cortical cell stiffness is independent of substrate mechanics. Nat. Mater. 19, 1019–1025 (2020).

    ADS  Google Scholar 

  223. Gavara, N. & Chadwick, R. S. Determination of the elastic moduli of thin samples and adherent cells using conical atomic force microscope tips. Nat. Nanotech 7, 733–736 (2012).

    ADS  Google Scholar 

  224. Harris, A. R. & Charras, G. T. Experimental validation of atomic force microscopy-based cell elasticity measurements. Nanotechnology 22, 345102 (2011).

    Google Scholar 

  225. Staunton, J. R., Doss, B. L., Lindsay, S. & Ros, R. Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices. Sci. Rep. 6, 19686 (2016).

    ADS  Google Scholar 

  226. Hobson, C. M. et al. Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics. Mol. Biol. Cell 31, 1788–1801 (2020).

    Google Scholar 

  227. Rico, F. et al. Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips. Phys. Rev. E Stat. Nonlin Soft Matter Phys 72, 021914 (2005).

    Google Scholar 

  228. Xiao, J. & Dufrêne, Y. F. Optical and force nanoscopy in microbiology. Nat. Microbiol. 1, 16186 (2016).

    Google Scholar 

  229. Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21, 72–84 (2019).

    Google Scholar 

  230. Miranda, A. et al. How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology? Nanoscale 13, 2082–2099 (2021).

    Google Scholar 

  231. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    ADS  Google Scholar 

  232. Hell, S. W. Microscopy and its focal switch. Nat. Methods 6, 24–32 (2009).

    Google Scholar 

  233. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    ADS  Google Scholar 

  234. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Google Scholar 

  235. Neil, M. A., Juskaitis, R. & Wilson, T. Method of obtaining optical sectioning by using structured light in a conventional microscope. Opt. Lett. 22, 1905–1907 (1997).

    ADS  Google Scholar 

  236. Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).

    ADS  Google Scholar 

  237. Odermatt, P. D. et al. High-resolution correlative microscopy: bridging the gap between single molecule localization microscopy and atomic force microscopy. Nano Lett. 15, 4896–4904 (2015).

    ADS  Google Scholar 

  238. Zhao, W. et al. Studying the nucleated mammalian cell membrane by single molecule approaches. PLoS ONE 9, e91595 (2014).

    ADS  Google Scholar 

  239. Gómez-Varela, A. I. et al. Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences. Sci. Rep. 10, 1122 (2020).

    ADS  Google Scholar 

  240. Harke, B., Chacko, J. V., Haschke, H., Canale, C. & Diaspro, A. A novel nanoscopic tool by combining AFM with STED microscopy. Optical Nanoscopy 1, 3 (2012).

    Google Scholar 

  241. Chacko, J. V., Zanacchi, F. C. & Diaspro, A. Probing cytoskeletal structures by coupling optical superresolution and AFM techniques for a correlative approach. Cytoskeleton 70, 729–740 (2013).

    Google Scholar 

  242. Curry, N., Ghézali, G., Kaminski Schierle, G. S., Rouach, N. & Kaminski, C. F. Correlative STED and atomic force microscopy on live astrocytes reveals plasticity of cytoskeletal structure and membrane physical properties during polarized migration. Front. Cell Neurosci. 11, 104 (2017).

    Google Scholar 

  243. Rankl, C., Kienberger, F., Gruber, H., Blaas, D. & Hinterdorfer, P. Accuracy estimation in force spectroscopy experiments. Jpn. J. Appl. Phys. 46, 5536–5539 (2007).

    ADS  Google Scholar 

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Acknowledgements

Work at the Université Catholique de Louvain was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 693630), the National Fund for Scientific Research (FNRS) and the Research Department of the Communauté française de Belgique (Concerted Research Action). Work at the Johannes Kepler University Linz was supported by the Austrian Science Fund (FWF) projects I 3173 (P.H. and Y.J.O.) and V584-BBL (Y.J.O.), and the Austrian National Foundation for Research, Technology, and Development and Research Department of the State of Upper Austria (Y.J.O.). D.A. and Y.F.D. are Research Associate and Research Director at the FNRS, respectively. The work at ETH Zurich was supported by the ETH Zurich (grant no. ETH-20 17-2), the Swiss National Science Foundation (grant no. 31003A_182587/1) and the NCCR Molecular Systems Engineering.

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Authors and Affiliations

  1. Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

    Albertus Viljoen, Marion Mathelié-Guinlet, Ankita Ray, David Alsteens & Yves F. Dufrêne

  2. Departement of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

    Nico Strohmeyer & Daniel J. Müller

  3. Institute of Biophysics, Johannes Kepler University, Linz, Austria

    Yoo Jin Oh & Peter Hinterdorfer

Authors
  1. Albertus Viljoen
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  2. Marion Mathelié-Guinlet
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  3. Ankita Ray
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  4. Nico Strohmeyer
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  5. Yoo Jin Oh
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  6. Peter Hinterdorfer
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  7. Daniel J. Müller
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  8. David Alsteens
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  9. Yves F. Dufrêne
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Contributions

Introduction (A.V., M.M.-G., A.R., N.S., Y.J.O., P.H., D.J.M., D.A. and Y.F.D.); Experimentation (A.V., M.M.-G., A.R., N.S., Y.J.O., P.H., D.J.M., D.A. and Y.F.D.); Results (A.V., M.M.-G., A.R., N.S., Y.J.O., P.H., D.J.M., D.A. and Y.F.D.); Applications (A.V., M.M.-G., A.R., N.S., Y.J.O., P.H., D.J.M., D.A. and Y.F.D.); Reproducibility and data deposition (A.V., M.M.-G., A.R., N.S., Y.J.O., P.H., D.J.M., D.A. and Y.F.D.); Limitations and optimizations (A.V., M.M.-G., A.R., N.S., Y.J.O., P.H., D.J.M., D.A. and Y.F.D.); Outlook (A.V., M.M.-G., A.R., N.S., Y.J.O., P.H., D.J.M., D.A. and Y.F.D.); Overview of the Primer (Y.F.D.).

Corresponding authors

Correspondence to Peter Hinterdorfer, Daniel J. Müller, David Alsteens or Yves F. Dufrêne.

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The authors declare no competing interests.

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Nature Reviews Methods Primers thanks C. Heu, S. Kasas, O. Sahin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Glossary

Energy landscape

A description in two or three dimensions of the energetic and kinetic properties of a specific bond, for example, receptor–ligand bonds

Cellular adhesion force

The force cells withstand before detaching from surfaces, for example, from extracellular matrix (ECM) proteins, other cells or substrates.

Cortical elasticity

The elasticity of the actomyosin cortex of a mammalian cell.

Cantilever deflection

Bending of the cantilever in response to forces applied to the cantilever.

Cognate ligands

Ligands specifically binding to a receptor.

Overtones

Waves of frequency that are a positive integer multiple of the frequency of the original wave.

Wave shapes

Oscillations of the cantilever.

Phase shift

A shift in the phase of an oscillation’s sin wavefunction.

Binding probability of the molecular interaction

The ratio between the number of force–distance curves displaying a specific rupture event (or events) and the total number of force–distance curves recorded.

Measurement frequency range

The range of resonance frequency operation.

Dynamic force spectroscopy

(DFS). An experiment where arrays of force–distance curves are recorded at varying pulling speeds.

Catch bonds

Specific bonds that increase lifetime with increasing force stressing them.

Contour length

(Lc). The length of the linearly extended molecule without stretching the polymer backbone.

Persistence length

(lp). A quantitative measure of a polymer’s elasticity.

Adhesins

Adhesion proteins on bacterial cell surfaces.

Slip bonds

Specific bonds that decrease lifetime with increasing force stressing them.

Integrins

A family of cell eukaryotic surface adhesion receptors that bind mainly to extracellular matrix (ECM) proteins.

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Viljoen, A., Mathelié-Guinlet, M., Ray, A. et al. Force spectroscopy of single cells using atomic force microscopy. Nat Rev Methods Primers 1, 63 (2021). https://doi.org/10.1038/s43586-021-00062-x

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