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Scanning angle interference microscopy reveals cell dynamics at the nanoscale

Nature Methods volume 9, pages 825–827 (2012)Cite this article

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Abstract

Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.

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Figure 1: Scanning angle interference microscopy.
Figure 2: Scanning angle interference imaging of microtubules and adhesion complexes.
Figure 3: Nanoscale dynamics of adhesion proteins in migrating cells.

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Acknowledgements

We thank J. Lakins for guidance on the construction of talin fusion constructs, T. Onuta for assistance with the fabrication of silicon wafers at the Maryland Nanocenter FabLab, D. Trono (École Polytechnique Fédérale de Lausanne) for the gift of second-generation lentiviral vectors, W. Hillen and H. Bujard (University of Heidelberg) for the gift of the rtTAs-M2 construct and M. Krummel and S. Peck for helpful discussions. Images for this study were acquired at the Nikon Imaging Center at the University of California–San Francisco. This work was supported by the Breast Cancer Research Program Department of Defense Era of Hope grant W81XWH-05-1-0330, US National Institutes of Health (NIH)/National Cancer Institute (NCI) grant 1U54CA163155-01, NCI grant U54CA143836-01 and NIH/NCI R01 CA138818-01A1.

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Authors and Affiliations

  1. Deparment of Surgery and Center for Bioengineering and Tissue Regeneration, University of California–San Francisco, San Francisco, California, USA

    Matthew J Paszek, Christopher C DuFort, Matthew G Rubashkin & Valerie M Weaver

  2. Bay Area Physical Sciences–Oncology Center, University of California–Berkeley, Berkeley, California, USA

    Matthew J Paszek, Christopher C DuFort, Matthew G Rubashkin, Jan T Liphardt & Valerie M Weaver

  3. National High Magnetic Field Laboratory, The Florida State University, Tallahassee, Florida, USA

    Michael W Davidson

  4. Department of Biological Sciences, The Florida State University, Tallahassee, Florida, USA

    Michael W Davidson

  5. Department of Biochemistry and Biophysics, University of California–San Francisco, San Francisco, California, USA

    Kurt S Thorn

  6. Department of Physics, University of California–Berkeley, Berkeley, California, USA

    Jan T Liphardt

  7. QB3–California Institute for Quantitative Biosciences, University of California–Berkeley, Berkeley, California, USA

    Jan T Liphardt

  8. Department of Anatomy, University of California–San Francisco, San Francisco, California, USA

    Valerie M Weaver

  9. Department of Bioengineering and Therapeutic Sciences, University of California–San Francisco, San Francisco, California, USA

    Valerie M Weaver

  10. Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California–San Francisco, San Francisco, California, USA

    Valerie M Weaver

  11. Helen Diller Family Comprehensive Cancer Center, University of California–San Francisco, San Francisco, California, USA

    Valerie M Weaver

Authors
  1. Matthew J Paszek
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  2. Christopher C DuFort
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  3. Matthew G Rubashkin
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  4. Michael W Davidson
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  5. Kurt S Thorn
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  6. Jan T Liphardt
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  7. Valerie M Weaver
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Contributions

M.J.P. and V.M.W. conceived and initiated the project. M.J.P. and K.S.T. designed the instrumentation. M.J.P., C.C.D. and M.G.R. designed and performed experiments. M.W.D. designed fluorescent protein constructs. M.J.P. and M.G.R. wrote the analysis software. V.M.W. and J.T.L. supervised the project. All authors wrote the paper.

Corresponding author

Correspondence to Valerie M Weaver.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 and Supplementary Note (PDF 3005 kb)

Supplementary Video 1

Paxillin and vinculin heights in adhesion complexes of migrating cells. Epifluorescence images (top) and SAIM 3D reconstructions (bottom) of paxillin-mEmerald and vinculin-mCherry in migrating epithelial cells. (AVI 6375 kb)

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Paszek, M., DuFort, C., Rubashkin, M. et al. Scanning angle interference microscopy reveals cell dynamics at the nanoscale. Nat Methods 9, 825–827 (2012). https://doi.org/10.1038/nmeth.2077

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