Optimized localization analysis for single-molecule tracking and super-resolution microscopy (original) (raw)

Nature Methods volume 7, pages 377–381 (2010)Cite this article

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Abstract

We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.

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References

  1. Born, M. & Wolf, E. Principles of Optics (Cambridge University Press, New York, 1999).
  2. Barak, L.S. & Webb, W.W. J. Cell Biol. 95, 846–852 (1982).
    Article CAS Google Scholar
  3. Yildiz, A. et al. Science 300, 2061–2065 (2003).
    Article CAS Google Scholar
  4. Okten, Z., Churchman, L.S., Rock, R.S. & Spudich, J.A. Nat. Struct. Mol. Biol. 11, 884–887 (2004).
    Article Google Scholar
  5. Betzig, E. et al. Science 313, 1642–1645 (2006).
    Article CAS Google Scholar
  6. Moerner, W.E. Proc. Natl. Acad. Sci. USA 104, 12596–12602 (2007).
    Article CAS Google Scholar
  7. Abraham, A.V., Ram, S., Chao, J., Ward, E.S. & Ober, R.J. Opt. Express 17, 23352–23373 (2010).
    Article Google Scholar
  8. Rao, C.R. Linear Statistical Inference and Its Applications (Wiley, New York, New York, 1973).
  9. Ober, R.J., Ram, S. & Ward, E.S. Biophys. J. 86, 1185–1200 (2004).
    Article CAS Google Scholar
  10. Robbins, M.S. & Hadwen, B.J. IEEE Trans. Electron. Dev. 50, 1227–1232 (2003).
    Article Google Scholar
  11. Enderlein, J., Toprak, E. & Selvin, P. Opt. Express 14, 8111–8120 (2006).
    Article CAS Google Scholar
  12. Forkey, J.N., Quinlan, M.E., Shaw, M.A., Corrie, J.E.T. & Goldman, Y.E. Nature 422, 399–404 (2003).
    Article CAS Google Scholar
  13. Toprak, E. et al. Proc. Natl. Acad. Sci. USA 103, 6495–6499 (2006).
    Article CAS Google Scholar
  14. Aguet, F., Geissbühler, S., Märki, I., Lasser, T. & Unser, M. Opt. Express 17, 6829–6848 (2009).
    Article CAS Google Scholar
  15. Toprak, E. & Selvin, P.R. Annu. Rev. Biophys. Biomol. Struct. 36, 349–369 (2007).
    Article CAS Google Scholar
  16. Thompson, R.E., Larson, D.R. & Webb, W.W. Biophys. J. 82, 2775–2783 (2002).
    Article CAS Google Scholar
  17. Bobroff, N. Rev. Sci. Instrum. 57, 1152–1157 (1986).
    Article Google Scholar
  18. Carter, A.R. et al. Appl. Opt. 46, 421–427 (2007).
    Article Google Scholar
  19. Berg-Sørensen, K. & Flyvbjerg, H. Rev. Sci. Instrum. 75, 594–612 (2004).
    Article Google Scholar
  20. Axelrod, D., Burghardt, T.P. & Thompson, N.L. Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).
    Article CAS Google Scholar
  21. Ulbrich, M.H. & Isacoff, E.Y. Nat. Methods 4, 319–321 (2007).
    Article CAS Google Scholar
  22. Churchman, L.S., Flyvbjerg, H. & Spudich, J.A. Biophys. J. 90, 668–671 (2006).
    Article CAS Google Scholar

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Acknowledgements

We thank S.M. Block, W.E. Moerner and R.S. Rock for discussions; Z.D. Bryant for allowing us to use his microscope for some of the data collection and M.W. Elting and J.M. Sung for assisting us. This work was supported by the European Union (FP7-HEALTH-F4-2008-201418, Revolutionary Approaches and Devices for Nucleic Acid Analysis to H.F.), by the US National Institutes of Health (GM33289 to L.S.C. and J.A.S.), by the Human Frontier Science Program (GP0054/2009-C to J.A.S. and H.F.) and the Damon Runyon Cancer Research Foundation (DRG-1997-08 to L.S.C.).

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Author notes

  1. Kim I Mortensen and L Stirling Churchman: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark
    Kim I Mortensen & Henrik Flyvbjerg
  2. Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA
    L Stirling Churchman & James A Spudich
  3. Department of Physics, Stanford University School of Medicine, Stanford, California, USA
    L Stirling Churchman

Authors

  1. Kim I Mortensen
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  2. L Stirling Churchman
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  3. James A Spudich
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  4. Henrik Flyvbjerg
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Contributions

H.F., K.I.M. and L.S.C. designed research; K.I.M. and H.F. performed the theoretical calculations and analyzed data; J.A.S. supervised the experiments; L.S.C. conducted experiments; K.I.M. did numerical simulations; H.F., K.I.M., L.S.C. and J.A.S. wrote the paper.

Corresponding author

Correspondence toHenrik Flyvbjerg.

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The authors declare no competing financial interests.

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Mortensen, K., Churchman, L., Spudich, J. et al. Optimized localization analysis for single-molecule tracking and super-resolution microscopy.Nat Methods 7, 377–381 (2010). https://doi.org/10.1038/nmeth.1447

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