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Micro and Nano Technologies in Bioanalysis pp 451–460Cite as

Single-Molecule Imaging of Fluorescent Proteins Expressed in Living Cells

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 544))

Summary

This chapter focuses on single-molecule imaging (SMI) in living cells using green fluorescent protein (GFP) or its related fluorescent protein tags (GFPs). Use of GFPs is a convenient technique to achieve molecular imaging of most proteins in living cells. However, because of difficulties in preparing samples suitable for SMI and the instability of fluorescence signals, special care is required for SMI using GFPs in living cells. Techniques for vector preparation, protein expression, sample preparation, microscopy, and image processing for SMI of GFPs in living cells are discussed in this chapter, along with examples of imaging applications. Double labeling of single molecules and single-pair fluorescent resonance energy transfer (spFRET) are possible in living cells using GFP and YFP as fluorescent tags. The limitations of SMI using GFPs are also discussed.

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References

  1. Funatsu, T., Harada, Y., Tokunaga, M., Saito, K., and Yanagida, T. (1995) Imaging of single fluorescent molecules and individual ATP turnover by single myosin molecules in aqueous solution. Nature 374, 555–559.

    Article  CAS  Google Scholar 

  2. Sase, I., Miyata, H., John, C. E. T., James, C. S., and Kinosita, K. Jr. (1995) Real time imaging of single fluorophores on moving actin with an epifluorescence microscope. Biophys. J. 69, 323–328.

    Article  CAS  Google Scholar 

  3. Cornish, P. and Ha, T. (2006) A survey of single molecule techniques in chemical biology. ACS Chem. Biol. 2, 53–61.

    Article  Google Scholar 

  4. Sako, Y. and Yanagida, T. (2003) Single-molecule visualization in cell biology. Nature Rev. Mol. Cell Biol. 4, SS1-5.

    Google Scholar 

  5. Sako, Y. (2006) Imaging single molecules for systems biology. Mol. Syst. Biol. doi:10.1038/msb4100100.

    Google Scholar 

  6. Sako, Y., Hibino, K., Miyauchi, T., Miyamoto, Y., Ueda, M., and Yanagida, T. (2000) Single-molecule imaging of signaling molecules in living cells. Single Mol. 1, 151–155.

    Article  Google Scholar 

  7. Tsien, R. Y. (2005) Building and breeding molecules to spy on cells and tumors. FEBS Lett. 579, 927–932.

    Article  CAS  Google Scholar 

  8. Kozak, M. (1999) Initiation of translocation in prokaryotes and eukaryotes. Gene 234,187–208.

    Article  CAS  Google Scholar 

  9. Zacharias, D. A., Violin, J. D., Newton, A. C., and Tsien, R. Y. (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916.

    Article  CAS  Google Scholar 

  10. Axelrod, D. (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774.

    Article  CAS  Google Scholar 

  11. Hibino, K., Watanabe, T., Kozuka, J., Iwane, A. H., Okada, T., Kataoka, T., Yanagida, T., and Sako, Y. (2003) Single- and multiple-molecule dynamics of the signaling from H-Ras to c-Raf1 visualized on the plasma membrane of living cells. Chem. Phys. Chem. 4, 748–753.

    Article  CAS  Google Scholar 

  12. Kusumi, A., Sako, Y., and Yamamoto, M. (1993) Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys. J. 65, 2021–2040.

    Article  CAS  Google Scholar 

  13. Xie, S. (2001) Single-molecule approach to enzymology. Single Mol. 2, 229–236.

    Article  CAS  Google Scholar 

  14. Kinosita K., Ito, H., Ishiwata, S., Hirano, K., Nishizaka, T., and Hayakawa, T (1991) Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium. J. Cell Biol. 115, 67–73.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Cellular Informatics Laboratory, 2-1 Hirosawa, 351-0198, RIKEN, Wako, Japan

    Kayo Hibino, Michio Hiroshima, Masahiro Takahashi & Yasushi Sako

Authors
  1. Kayo Hibino
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  2. Michio Hiroshima
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  3. Masahiro Takahashi
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  4. Yasushi Sako
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Corresponding author

Correspondence to Yasushi Sako .

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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Hibino, K., Hiroshima, M., Takahashi, M., Sako, Y. (2009). Single-Molecule Imaging of Fluorescent Proteins Expressed in Living Cells. In: Foote, R., Lee, J. (eds) Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology™, vol 544. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-483-4_30

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    • DOI: https://doi.org/10.1007/978-1-59745-483-4_30

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    • Publisher Name: Humana Press, Totowa, NJ

    • Print ISBN: 978-1-934115-40-4

    • Online ISBN: 978-1-59745-483-4

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