Skip to main content

Analysis of Human Fibroblasts by Atomic Force Microscopy

  • Protocol
  • 1464 Accesses

Part of the Methods in Molecular Biology™ book series (MIMB,volume 242)

Abstract

The force-sensing members of the large family of scanning probe microscopies have become important tools during the past decade for visualizing, characterizing, and manipulating objects and processes on the meso- and nanoscale level. The atomic force microscope (AFM), in particular, has had an impact in the life sciences. In cell science, the pioneering work with AFM was conducted in the early 1990s (13). The methodologies have now reached a stage of relative maturity (4). The principal merit of the AFM is as a nonintrusive local probe of live cells and their dynamics in the biofluid environment. As well as offering high spatial resolution imaging in one or more operational modes, the AFM can deliver characterization of mechanical properties and local chemistry through operation in the force-vs-distance (F-d) mode (e.g., ref. 5). The lateral resolution delivered by the AFM will in most cases, and especially for soft materials, be inferior to that obtained by electron-optical techniques, but the z-resolution is routinely in the nanometer range with a depth of focus equal to the dynamic range of the z-stage travel. The instrument may be operated in one of several modes, of which the most common ones are as follows: the contact mode, using a soft lever in which contours of constant strength of interaction are traced out; the intermittent-contact mode, in which a relatively stiff lever is vibrated at a frequency near that of a free-running resonance and in which contours of constant decrement of the free-running amplitude or a constant phase shift are mapped; and the F-d mode, in which the local stiffness of interaction between tip and specimen is determined over a range of applied force (lever deflection and z-stage travel being the two measurable variables).

Keywords

  • Atomic Force Microscope
  • Scanning Probe Microscopy
  • Atomic Force Microscope Analysis
  • High Spatial Resolution Imaging
  • Constant Phase Shift

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1385/1-59259-647-9:53
  • Chapter length: 15 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-59259-647-8
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   149.99
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3.
Fig. 4.
Fig. 5.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Gould, S. A. C., Drake, B., Prater, C. B., Weisenhorn, A. L., Manne, S., Hansma, H. G., et al. (1990) From atoms to integrated-circuit chips, blood-cells, and bacteria with the atomic force microscope. J. Vac. Sci. Technol. A 8, 369–373.

    CrossRef  CAS  Google Scholar 

  2. Henderson, E., Haydon, P. G., and Sakaguchi, D. S. (1992) Actin filament dynamics in living glial cells imaged by atomic force microscopy. Science 257, 1944–1946.

    PubMed  CrossRef  CAS  Google Scholar 

  3. Hoh, J. H. and Hansma, P. K. (1992) Atomic force microscopy for high-resolution imaging in cell biology. Trends Cell Biol. 2, 208–212.

    PubMed  CrossRef  CAS  Google Scholar 

  4. Hong, X. and Lei, Y. (1999) Atomic force microscopy of living cells: progress, problems and prospects. Methods Cell Sci. 21, 1–17.

    Google Scholar 

  5. Bushell, G. R., Cahill, C., Clarke, F. M, Gibson, C. T., Myhra, S., and Watson, G. S. (1999) Imaging and force-distance analysis of human fibroblasts in vitro by atomic force microscopy. Cytometry 36, 254–264.

    PubMed  CrossRef  CAS  Google Scholar 

  6. Pietrasanta, L. I., Schaper, A., and Jovin, T. M. (1994) Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy. J. Cell Sci. 107, 2427–2437.

    PubMed  Google Scholar 

  7. Gibson, C. T., Watson, G. S., and Myhra, S. (1996) Determination of the spring constants of probes for force microscopy/spectroscopy. Nano-technology 7, 259–262.

    Google Scholar 

  8. Gibson, C. T., Watson, G. S., and Myhra, S. (1997) Scanning force microscopy-calibrative procedures for ‘best practice’. Scanning 19, 564–581.

    CrossRef  Google Scholar 

  9. Putman, C. A. J., van der Werf, K. O., de Grooth, B. G., van Hulst, N. F., and Greve, J. (1994) Viscoelsticity of living cells allows high resolution imaging by tapping mode atomic force microscopy. Biophys. J. 67,1749–1753.

    PubMed  CrossRef  CAS  Google Scholar 

  10. Le Grimellec, C., Lesniewska, E., Giocondi, M.-C., Finot, E., and Goudonnet, J.-P. (1997) Simultaneous imaging of the surface and submembraneous cytoskeleton hi living cells by tapping mode atomic force microscopy. Acad. Sci. Biophys. 320, 637–643.

    Google Scholar 

  11. Vie, V., Giocondi, M.-C., Lesniewska, E., Finot, E., Goudonnet, J.-P., and Le Grimellec, C. (2000) Tapping-mode atomic force microscopy on intact cells: optimal adjustment of tapping conditions by using the dflection signal. Ultramicroscopy 82, 279–288.

    PubMed  CrossRef  CAS  Google Scholar 

  12. Schoenenberger, C.-A., and Hoh, J. H. (1994) Slow cellular dynamics in MDCK and R5 cells monitored by time-lapse atomic force microscopy. Biophys. J. 67, 929–936.

    PubMed  CrossRef  CAS  Google Scholar 

  13. Braet, F., Saynaeve, C., de Zanger, R., and Wisse, E. (1998) Imaging surface and submembraneous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages. J. Microsc. 190, 328–338.

    PubMed  CrossRef  CAS  Google Scholar 

  14. Rotsch, C. and Radmacher, M. (2000) Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys. J. 78, 520–535.

    PubMed  CrossRef  CAS  Google Scholar 

  15. Shroff, S. G., Saner, D. R., and Lai, R. (1995) Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy. Am. J. Physiol. 269, C286–C292.

    PubMed  CAS  Google Scholar 

  16. Domke, J., Parak, W. J., George, M., Gaub, H. E., and Radmacher, M. (1999) Mapping the mechanical pulse of single cardiomyocytes with the atomic force microscope. Eur. Biophys. J. 28,179–186.

    PubMed  CrossRef  CAS  Google Scholar 

  17. Crossley, J. A. A., Gibson, C. T., Mapledoram, L. D., Huson, M. G., Myhra, S., Pham, D. K., et al. (2000) Atomic force microscopy analysis of wool fibre surfaces in air and under water. Micron 31, 659–667.

    PubMed  CrossRef  Google Scholar 

  18. Blach, J., Loughlin, W., Watson, G., and Myhra, S. (2001) Surface characterization of human hair by atomic force microscopy in the imaging and F-d modes. Int. J. Cosm. Sci. 23,165–174.

    CrossRef  CAS  Google Scholar 

  19. Wu, H. W., Kuhn, T., and Moy, V. T. (1998) Mechanical properties of L929 cells measured by atomic force microscopy: effects of anticytoskeletal drugs and membrane crosslinking. Scanning 20, 389–397.

    PubMed  CrossRef  CAS  Google Scholar 

  20. Kuznetsov, Y. G., Malkin, A. J., and McPherson, A. (1997) Atomic force microscopy studies of living cells: Visualization of motility, division, aggregation, transformation and apoptosis. J. Struct. Biol. 120,180–191.

    PubMed  CrossRef  CAS  Google Scholar 

  21. Wu, H. W., Kuhn, T., and Moy, V. T. (1998) Mechanical properties of L929 cells measured by atomic force microscopy: effects of anticytoskeletal drugs and membrane crosslinking. Scanning 20, 389–397.

    PubMed  CrossRef  CAS  Google Scholar 

  22. Rotsch, C., Jacobson, K., and Radmacher, M. (1999) Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy. Proc. Natl. Acad. Sci. USA 96, 921–926.

    PubMed  CrossRef  CAS  Google Scholar 

  23. Ricci, D., Tedesco, M., and Grattarola, M. (1997) Mechanical and morphological properties of living 3T6 cells probed via scanning force microscopy. Microsc. Res. Tech. 36, 165–171.

    PubMed  CrossRef  CAS  Google Scholar 

  24. Haga, H., Sasaki, S., Kawabata, K., Ito, E., Ushiki, T., and Sambongi, T. (2000) Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton. Ultramicroscopy 82, 253–258.

    PubMed  CrossRef  CAS  Google Scholar 

  25. Haga, H., Nagayama, M., Kawabata, K., Ito, E., Ushiki, T., and Sambongi, T. (2000) Time-lapse viscoelastic imaging of living fibroblasts using force modulation in AFM. J. Electron Microsc. 49, 473–481.

    CAS  Google Scholar 

  26. Hellemans, L., Waeyaert, K., and Hennau, F. (1991) Can atomic force microscopy tips be inspected by atomic force microscopy? J. Vac. Sci. Technol. B9, 1309–1312.

    CrossRef  CAS  Google Scholar 

  27. Bushell, G. R., Watson, G. S., Holt, S.A., and Myhra, S. (1995) Imaging and nano-dissection of tobacco mosaic virus by atomic force microscopy. J. Microsc. 180,174–181.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Bushell, G.R., Cahill, C., Myhra, S., Watson, G.S. (2004). Analysis of Human Fibroblasts by Atomic Force Microscopy. In: Braga, P.C., Ricci, D. (eds) Atomic Force Microscopy. Methods in Molecular Biology™, vol 242. Humana Press. https://doi.org/10.1385/1-59259-647-9:53

Download citation

  • DOI: https://doi.org/10.1385/1-59259-647-9:53

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-094-6

  • Online ISBN: 978-1-59259-647-8

  • eBook Packages: Springer Protocols