Fluorescence Imaging with One Nanometer Accuracy: In Vitro and In Vivo Studies of Molecular Motors

  • Melinda Tonks Hoffman
  • Janet Sheung
  • Paul R. SelvinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 778)


Traditional microscopy techniques are limited by the wave-like characteristics of light, which dictate that about 250 nm (or roughly half the wavelength of the light) is the smallest distance by which two identical objects can be separated while still being able to distinguish between them. Since most biological molecules are much smaller than this limit, traditional light microscopes are generally not sufficient for single-molecule biological studies. Fluorescence Imaging with One Nanometer Accuracy (FIONA) is a technique that makes possible localization of an object to approximately one nanometer. The FIONA technique is simple in concept; it is built upon the idea that, if enough photons are collected, one can find the exact center of a fluorophore’s emission to within a single nanometer and track its motion with a very high level of precision. The center can be localized to approximately (λ/2)/Ö—N, where λ is the wavelength of the light and N is the number of photons collected. When N  =  10,000, FIONA achieves an accuracy of 1–2 nm, assuming the background is sufficiently low. FIONA, thus, works best with the use of high-quality dyes and fluorescence stabilization buffers, sensitive detection methods, and special microscopy techniques to reduce background fluorescence. FIONA is particularly well suited to the study of molecular motors, which are enzymes that couple ATP hydrolysis to conformational change and motion. In this chapter, we discuss the practical application of FIONA to molecular motors or other enzymes in biological systems.

Key words

FIONA Molecular motors Single-molecule tracking TIRF microscopy 



The authors acknowledge the NIH and NSF for financial support.


  1. 1.
    Huang, B., Bates, M., Huang, X. (2009) Super resolution fluorescence microscopy. Ann. Rev. Biochem. 78, 993–1016.PubMedCrossRefGoogle Scholar
  2. 2.
    Hell, S. W. (2007) Far-Field Optical Nanoscopy. Science 316, 1153–1158.PubMedCrossRefGoogle Scholar
  3. 3.
    Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., Goldman, Y. E., Selvin, P. R. (2003) Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization. Science 300, 2061–2065.PubMedCrossRefGoogle Scholar
  4. 4.
    Kural, C., Kim, H., Syed, S., Goshima, G., Gelfand, V. I., Selvin, P. R. (2005) Kinesin & Dynein Move a Peroxisome In Vivo: A Tug-of-War or Coordinated Movement? Science 308, 1469–1472.PubMedCrossRefGoogle Scholar
  5. 5.
    Thomson, R. E., Larson, D. R., Webb, W. W. (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783.CrossRefGoogle Scholar
  6. 6.
    Enderlein, J., Toprak, E., Selvin, P. R. (2006) Polarization effect on position accuracy of fluorphore localization. Opt. Express 14, 8111–8120.PubMedCrossRefGoogle Scholar
  7. 7.
    Selvin, P. R., Lougheed, T., Hoffman, M. T., Park, H., Balci, H., Blehm, B. H., Toprak, E. (2008) In vitro and in vivo FIONA and other acronyms for watching molecular motors walk. In Selvin PR, H. T., ed.: Single-Molecule Techniques. Cold Spring Harbor Laboratory Press, Cold Spring Harbor 37–71.Google Scholar
  8. 8.
    Pierce, D. W., Vale, R. D. (1998) Assaying Processive Movement of Kinesin by Fluorescence Microscopy. Methods Enzymol. 298, 154–171.PubMedCrossRefGoogle Scholar
  9. 9.
    Ozeki, T., Verma, V., Uppalapti, M., Suzuki, Y., Nakamura, M., Catchmark, J., Hancock, W. O. (2009) Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 Surfaces. Biophys. J. 96, 3305–3318.PubMedCrossRefGoogle Scholar
  10. 10.
    Aitken, C. E., Marshall, R. A., Puglisi, J. D. (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835.PubMedCrossRefGoogle Scholar
  11. 11.
    Müllner, F. E., Syed, S., Selvin, P. R., Sigworth, F. J. (2010) Improved hidden Markov models for molecular motors.1. Basic theory. Biophys J.Google Scholar
  12. 12.
    Syed, S., Müllner, F., Selvin, P. R., Sigworth, F. J. (2010) Improved hidden Markov models for molecular motors. 2. Extensions and application to experimental data. Biophys. J.Google Scholar
  13. 13.
    Rasnik, I., McKinney, S. A., Ha, T. (2005) Surfaces and Orientations: Much to FRET about? Acc. Chem. Res. 38, 542–548.PubMedCrossRefGoogle Scholar
  14. 14.
    Kural, C., Serpinskaya, A. S., Chou, Y. H., Goldman, R. D., Gelfand, V. I. (2007) Tracking melanosomes inside a cell to study molecular motors and their interaction. PNAS 104, 5378–5382.PubMedCrossRefGoogle Scholar
  15. 15.
    Jablonski, A. E., Humphries, W. H., Payne, C. K. (2009) Pyrenebutyrate-Mediated Delivery of Quantum Dots across the Plasma Membrane of Living Cells. J. Phys. Chem. B, 405–408.Google Scholar
  16. 16.
    Nan, X., Sims, P. A., Chen, P., Xie, X. S. (2005) Observation of Individual Microtubule Motor Steps in Living Cells with Endocytosed Quantum Dots. J. Phys. Chem. Lett. 109, 24220–24224.Google Scholar
  17. 17.
    Courty, S., Luccardini, C., Bellaiche, Y. (2006) Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot Imaging. Nano Lett 6, 1491–1495.PubMedCrossRefGoogle Scholar
  18. 18.
    Derfus, A. M., Chan, W. C., Bhatia, S. N. (2004) Intracellular Delivery of Quantum Dots for Live Cell Labeling and Organelle Tracking. Adv Mater 16, 961–966.CrossRefGoogle Scholar
  19. 19.
    Chattopadhaya, S., Srinivasan, R., Yeo, D. S., Chen, G., Yao, S. Q. (2009) Site-specific covalent labeling of proteins inside live cells using small molecule probes. Bioorg. Med. Chem. Lett. 981–989.Google Scholar
  20. 20.
    Miller, L. W., Cornish, V. W. (2005) Selective chemical labeling of proteins in living cells. Curr. Opin. Chem. Biol. 9, 56–61.PubMedCrossRefGoogle Scholar
  21. 21.
    Rasnik, I., McKinney, S. M., Ha, T. (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods 3, 891–893.PubMedCrossRefGoogle Scholar
  22. 22.
    Chattopadhaya, S., Srinivasan, R., Yeo, D., Chen, G., Yao, S. (2009) Site-specific covalent labeling of proteins inside live cells using small molecule probes. Bioorg. Med. Chem. 17, 981–989.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Melinda Tonks Hoffman
    • 1
  • Janet Sheung
    • 1
  • Paul R. Selvin
    • 1
    Email author
  1. 1.Physics DepartmentUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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