Abstract

We measured the membrane topography and dynamics on a living fibroblast by using the non-interferometric widefield optical profilometry (NIWOP) technique. With a water-immersion objective of a 0.75 numerical aperture, our NIWOP system provides depth resolution about 20 nm. The imaging speed could be as high as 5 frames/min. We directly observed and profiled the inward propagation of membrane ripples near the cell edge. To verify if the membrane activity was driven by the underlying cytoskeleton, we changed the structure of the cell cortex while observing the membrane topography. After dissolving the actin cortex by cytochalasin D, we found that the propagation of the membrane ripples disappeared and the edge of the cell shank. The non-contact NIWOP technique does not affect the motility and viability of cells and therefore is suitable for the studies on cell physiology related to membrane motions.

© 2005 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |

  1. B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Molecular Biology of the Cell, 4th ed. (Garland Science, New York, 2002).
  2. H.-B. Wang, M. Dembo, S. K. Hanks, and Y.-L. Wang, "Focal adhesion kinase is involved in mechanosensing during fibroblast migration," Proc. Natl. Acad. Sci. USA 98, 11295-11300 (2001).
    [CrossRef] [PubMed]
  3. J. E. Bear, T. M. Syitkina, M. Krause, D. A. Schafer, J. J. Loureiro, G. A. Strasser, V. Maly, O. Y. Chaga, J. A. Cooper, G. G. Borisy, and F. B. Gertler, "Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility," Cell 109, 509-521 (2002).
    [CrossRef] [PubMed]
  4. B. D. Harms, G. M. Bassi, A. R. Horwitz, and D. A. Lauffenburger, "Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions," Biophys. J. 88, 1479-1488 (2005).
    [CrossRef] [PubMed]
  5. T. Akkin, D. P. Davé, T. E. Milner, and H. G. Rylander III, "Detection of neural activity using phase-sensitive optical low-coherence reflectometry," Opt. Express 12, 2377-2386 (2004). <a href= "http://www.opticsexpress.org/abstract.cfm?URI=OPEX-12-11-2377">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-12-11-2377</a>
    [CrossRef] [PubMed]
  6. C. Rotsch, K. Jacobson, and M. Radmacher, "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 (1999).
    [CrossRef] [PubMed]
  7. B. P. Jena and J. K. H. Horber, eds., Atomic Force Microscopy in Cell Biology, Methods in Cell Biology 68, (Academic Press, San Diego, 2002).
  8. R. E. Mahaffy, S. Park, E. Gerde, J. Kas, and C. K. Shih, "Quantitative analysis of the viscoelastic properties of thin regions of fibroblasts using atomic force microscopy," Biophys. J. 86, 1777-1793 (2004).
    [CrossRef] [PubMed]
  9. C.-H. Lee, H.-Y. Mong, and W.-C. Lin, "Noninterferometric wide-field optical profilometry with nanometer depth resolution," Opt. Lett. 27, 1773-1775 (2002).
    [CrossRef]
  10. M. A. A. Neil, R. Juskaitis, and T. Wilson, "Method of obtaining optical sectioning by using structured light in a conventional microscope," Opt. Lett. 22, 1905-1907 (1997).
    [CrossRef]
  11. S.-W. Huang, H.-Y. Mong, and C.-H. Lee, "Super-resolution bright-field optical microscopy based on nanometer topographic contrast," Microsc. Res. Tech. 65, 180-185 (2004).
    [CrossRef]
  12. C.-H. Lee, C.-L. Guo, and J. Wang, "Optical measurement of the viscoelastic and biochemical responses of living cells to mechanical perturbation," Opt. Lett. 23, 307-309 (1998).
    [CrossRef]
  13. A. Dubois, A. C. Boccara, and M. Lebec, "Real-time reflectivity and topography imagery of depth-resolved microscopic surfaces," Opt. Lett. 24, 309-311 (1999).
    [CrossRef]

Biophys. J. (2)

B. D. Harms, G. M. Bassi, A. R. Horwitz, and D. A. Lauffenburger, "Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions," Biophys. J. 88, 1479-1488 (2005).
[CrossRef] [PubMed]

R. E. Mahaffy, S. Park, E. Gerde, J. Kas, and C. K. Shih, "Quantitative analysis of the viscoelastic properties of thin regions of fibroblasts using atomic force microscopy," Biophys. J. 86, 1777-1793 (2004).
[CrossRef] [PubMed]

Cell (1)

J. E. Bear, T. M. Syitkina, M. Krause, D. A. Schafer, J. J. Loureiro, G. A. Strasser, V. Maly, O. Y. Chaga, J. A. Cooper, G. G. Borisy, and F. B. Gertler, "Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility," Cell 109, 509-521 (2002).
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

S.-W. Huang, H.-Y. Mong, and C.-H. Lee, "Super-resolution bright-field optical microscopy based on nanometer topographic contrast," Microsc. Res. Tech. 65, 180-185 (2004).
[CrossRef]

Opt. Express (1)

Opt. Lett. (4)

Proc. Natl. Acad. Sci. USA (2)

C. Rotsch, K. Jacobson, and M. Radmacher, "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 (1999).
[CrossRef] [PubMed]

H.-B. Wang, M. Dembo, S. K. Hanks, and Y.-L. Wang, "Focal adhesion kinase is involved in mechanosensing during fibroblast migration," Proc. Natl. Acad. Sci. USA 98, 11295-11300 (2001).
[CrossRef] [PubMed]

Other (2)

B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Molecular Biology of the Cell, 4th ed. (Garland Science, New York, 2002).

B. P. Jena and J. K. H. Horber, eds., Atomic Force Microscopy in Cell Biology, Methods in Cell Biology 68, (Academic Press, San Diego, 2002).

Supplementary Material (1)

» Media 1: AVI (2129 KB)     

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (6)

Fig. 1.
Fig. 1.

Setup of the non-interferometric widefield optical profilometer. The system is constructed on a microscope with epi-illumination.

Fig. 2.
Fig. 2.

Linear region of the axial response curve of our NIWOP system. Every dot and error bar represent the average value and standard deviation of intensity measured for 20 times at that axial position. The slope of the fitting line is 0.68 μm-1. The operational dynamic range is 1.0 μm.

Fig. 3.
Fig. 3.

(a) Bright field image of a living HS-68 fibroblast. (b) The raw NIWOP image of the same cell. The membrane ripples are visible, but overlapped with other optical contrast. (c) The NIWOP image after the processing described in the text. The gray scale in this image corresponds to height variations from 0 to 350 nm. Membrane ripples are much clearer.

Fig. 4.
Fig. 4.

Propagation of the membrane ripples on a fibroblast. Panels (a) to (e) are the zoom-in images of the region enclosed by the dashed rectangle in the whole-view image. An arrow indicates one of the ripples for a better visualization of the propagation. From (a) to (e) we see the ripples are moving away from the cell edge with an average speed about 1.3 μm/hour. The dynamics is more obvious in Fig. 5.

Fig. 5.
Fig. 5.

(2.1 MB) Movie of the membrane-ripple propagation. 20 μM cytochalasin D was added into the culture medium at 3:00.

Fig. 6.
Fig. 6.

Membrane profile of the same cell in Fig. 4 after the treatment of 20 μM cytochalasin D. The time interval between each image is 10 minutes. The dashed line indicates the boundary of the cell. (a) 10 minutes after the treatment. We see sudden arising of multiple peaks on the membrane. (b) The cell contracts and the membrane ripples become more random and smaller compared to those in (a). (c) 10 minutes later, the cell shape and membrane profile do not show significant variation. [Media 1]

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

I ( x , y ) = I m ( x , y ) α T m ( x , y ) + I d ( x , y ) α Z d ,
I B ( x , y ) = I m ( x , y ) + I d ( x , y ) ,

Metrics