Abstract

Measuring subdiffraction separations between single fluorescent particles is important for biological, nano-, and medical-technology studies. Major challenges include (i) measuring changing molecular separations with high temporal resolution while (ii) using identical fluorescent labels. Here we report a method that measures subdiffraction separations between two identical fluorophores by using a single image of milliseconds exposure time and a standard single-molecule fluorescent imaging setup. The fluorophores do not need to be bleached and the separations can be measured down to 40 nm with nanometer precision. The method is called single-molecule image deconvolution — SMID, and in this article it measures the standard deviation (SD) of Gaussian-approximated combined fluorescent intensity profiles of the two subdiffraction-separated fluorophores. This study enables measurements of (i) subdiffraction dimolecular separations using a single image, lifting the temporal resolution of seconds to milliseconds, while (ii) using identical fluorophores. The single-image nature of this dimer separation study makes it a single-image molecular analysis (SIMA) study.

©2010 Optical Society of America

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References

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  1. M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, UK, 1999).
  2. G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
    [Crossref] [PubMed]
  3. A. K. Salem, P. C. Searson, and K. W. Leong, “Multifunctional nanorods for gene delivery,” Nature 2, 668–671 (2003).
    [Crossref]
  4. G. Han, P. Ghosh, M. De, and V. M. Rotello, “Drug and gene delivery using gold nanoparticles,” Nanobiotechnology 3, 40–45 (2007).
    [Crossref]
  5. A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303, 676–678 (2004).
    [Crossref]
  6. X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).
    [Crossref] [PubMed]
  7. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
    [Crossref] [PubMed]
  8. A. Sharonov and R. M. Hochstrasser, “Wide-field subdiffraction imaging by accumulated binding of diffusing probes,” Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).
    [Crossref] [PubMed]
  9. T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
    [Crossref] [PubMed]
  10. L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
    [Crossref]
  11. M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science 317, 1749–1753 (2007).
    [Crossref] [PubMed]
  12. H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position,” Biophys. J. 67, 1291–1300 (2007).
    [Crossref]
  13. G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
    [Crossref]
  14. M. Speidel, A. Jonas, and E.-L. Florin, “Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging,” Opt. Lett. 28, 69–71 (2003).
    [Crossref] [PubMed]
  15. L Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90, 053902 (2007).
    [Crossref]
  16. D. A. Agard, R. A. Steinberg, and R. M. Stroud, “Quantitative analysis of electrophoretograms: A mathematical approach to super-resolution,” Anal. Chem. 111, 257–268 (1981).
  17. J. M. Smith and D. J. Thomas, “Quantitative analysis of one-dimensional gel electrophoresis profiles,” Comput. Appl. Biosci. 317, 1749–1753 (2007).
  18. J. Behboodian, “On the modes of a mixture of two normal distributions,” Technometrics 12, 131–139 (1970).
    [Crossref]
  19. M. C. DeSantis, S. H. DeCenzo, J. L. Li, and Y. M. Wang, “Precision analysis for standard deviation measurements of single-fluorescent molecule images,” Opt. Express 18, 6563–6576 (2010).
    [Crossref] [PubMed]
  20. R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775–2783 (2002).
    [Crossref] [PubMed]
  21. S. Ram, E. S. Ward, and R. J. Ober, “Beyond Rayleigh’s criterion: A resolution measure with application to single-molecule microscopy,” Proc. Natl. Acad. Sci. USA 103, 4457–4462 (2006).
    [Crossref] [PubMed]
  22. Y. M. Wang, R. H. Austin, and E. C. Cox, “Single molecule measurements of repressor protein 1D diffusion on DNA,” Phys. Rev. Lett. 97, 048302 (2006).
    [Crossref] [PubMed]
  23. Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
    [Crossref] [PubMed]

2010 (1)

2007 (5)

G. Han, P. Ghosh, M. De, and V. M. Rotello, “Drug and gene delivery using gold nanoparticles,” Nanobiotechnology 3, 40–45 (2007).
[Crossref]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position,” Biophys. J. 67, 1291–1300 (2007).
[Crossref]

L Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90, 053902 (2007).
[Crossref]

J. M. Smith and D. J. Thomas, “Quantitative analysis of one-dimensional gel electrophoresis profiles,” Comput. Appl. Biosci. 317, 1749–1753 (2007).

2006 (4)

S. Ram, E. S. Ward, and R. J. Ober, “Beyond Rayleigh’s criterion: A resolution measure with application to single-molecule microscopy,” Proc. Natl. Acad. Sci. USA 103, 4457–4462 (2006).
[Crossref] [PubMed]

Y. M. Wang, R. H. Austin, and E. C. Cox, “Single molecule measurements of repressor protein 1D diffusion on DNA,” Phys. Rev. Lett. 97, 048302 (2006).
[Crossref] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

A. Sharonov and R. M. Hochstrasser, “Wide-field subdiffraction imaging by accumulated binding of diffusing probes,” Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).
[Crossref] [PubMed]

2005 (2)

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
[Crossref]

2004 (2)

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303, 676–678 (2004).
[Crossref]

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).
[Crossref] [PubMed]

2003 (2)

2002 (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775–2783 (2002).
[Crossref] [PubMed]

2000 (2)

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

1997 (1)

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

1981 (1)

D. A. Agard, R. A. Steinberg, and R. M. Stroud, “Quantitative analysis of electrophoretograms: A mathematical approach to super-resolution,” Anal. Chem. 111, 257–268 (1981).

1970 (1)

J. Behboodian, “On the modes of a mixture of two normal distributions,” Technometrics 12, 131–139 (1970).
[Crossref]

Agard, D. A.

D. A. Agard, R. A. Steinberg, and R. M. Stroud, “Quantitative analysis of electrophoretograms: A mathematical approach to super-resolution,” Anal. Chem. 111, 257–268 (1981).

Alivisatos, A. P.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

Austin, R. H.

Y. M. Wang, R. H. Austin, and E. C. Cox, “Single molecule measurements of repressor protein 1D diffusion on DNA,” Phys. Rev. Lett. 97, 048302 (2006).
[Crossref] [PubMed]

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Bates, M.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

Behboodian, J.

J. Behboodian, “On the modes of a mixture of two normal distributions,” Technometrics 12, 131–139 (1970).
[Crossref]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Born, M.

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, UK, 1999).

Chemla, D. S.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

Churchman, L. S.

L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
[Crossref]

Cox, E. C.

Y. M. Wang, R. H. Austin, and E. C. Cox, “Single molecule measurements of repressor protein 1D diffusion on DNA,” Phys. Rev. Lett. 97, 048302 (2006).
[Crossref] [PubMed]

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Dawson, J. F.

L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
[Crossref]

De, M.

G. Han, P. Ghosh, M. De, and V. M. Rotello, “Drug and gene delivery using gold nanoparticles,” Nanobiotechnology 3, 40–45 (2007).
[Crossref]

DeCenzo, S. H.

Dempsey, G. T.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

DeSantis, M. C.

Florin, E.-L.

Ghosh, P.

G. Han, P. Ghosh, M. De, and V. M. Rotello, “Drug and gene delivery using gold nanoparticles,” Nanobiotechnology 3, 40–45 (2007).
[Crossref]

Golding, I.

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Gordon, G. S.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Gruber, H. J.

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

Guan, X.-J.

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Guo, L.

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Han, G.

G. Han, P. Ghosh, M. De, and V. M. Rotello, “Drug and gene delivery using gold nanoparticles,” Nanobiotechnology 3, 40–45 (2007).
[Crossref]

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Hochstrasser, R. M.

A. Sharonov and R. M. Hochstrasser, “Wide-field subdiffraction imaging by accumulated binding of diffusing probes,” Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).
[Crossref] [PubMed]

Holtzer, L

L Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90, 053902 (2007).
[Crossref]

Huang, B.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

Jonas, A.

Kao, H. P.

H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position,” Biophys. J. 67, 1291–1300 (2007).
[Crossref]

Knaus, H.

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

kten, Z.

L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
[Crossref]

Lacoste, T. D.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775–2783 (2002).
[Crossref] [PubMed]

Leong, K. W.

A. K. Salem, P. C. Searson, and K. W. Leong, “Multifunctional nanorods for gene delivery,” Nature 2, 668–671 (2003).
[Crossref]

Li, J. L.

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Losick, R.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Meckel, T.

L Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90, 053902 (2007).
[Crossref]

Mets, L.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).
[Crossref] [PubMed]

Michalet, X.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

Murray, A. W.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Ober, R. J.

S. Ram, E. S. Ward, and R. J. Ober, “Beyond Rayleigh’s criterion: A resolution measure with application to single-molecule microscopy,” Proc. Natl. Acad. Sci. USA 103, 4457–4462 (2006).
[Crossref] [PubMed]

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Pastushenko, V. Ph.

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Pinaud, F.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

Pragl, B.

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

Qu, X.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).
[Crossref] [PubMed]

Ram, S.

S. Ram, E. S. Ward, and R. J. Ober, “Beyond Rayleigh’s criterion: A resolution measure with application to single-molecule microscopy,” Proc. Natl. Acad. Sci. USA 103, 4457–4462 (2006).
[Crossref] [PubMed]

Reisner, W.

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Riehn, R.

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Rock, R. S.

L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
[Crossref]

Rotello, V. M.

G. Han, P. Ghosh, M. De, and V. M. Rotello, “Drug and gene delivery using gold nanoparticles,” Nanobiotechnology 3, 40–45 (2007).
[Crossref]

Salem, A. K.

A. K. Salem, P. C. Searson, and K. W. Leong, “Multifunctional nanorods for gene delivery,” Nature 2, 668–671 (2003).
[Crossref]

Scherer, N. F.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).
[Crossref] [PubMed]

Schindler, H.

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

Schmidt, T.

L Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90, 053902 (2007).
[Crossref]

Schtz, G. J.

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

Schwatz, J. L.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Searson, P. C.

A. K. Salem, P. C. Searson, and K. W. Leong, “Multifunctional nanorods for gene delivery,” Nature 2, 668–671 (2003).
[Crossref]

Selvin, P. R.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303, 676–678 (2004).
[Crossref]

Sharonov, A.

A. Sharonov and R. M. Hochstrasser, “Wide-field subdiffraction imaging by accumulated binding of diffusing probes,” Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).
[Crossref] [PubMed]

Sitnikov, D.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Smith, J. M.

J. M. Smith and D. J. Thomas, “Quantitative analysis of one-dimensional gel electrophoresis profiles,” Comput. Appl. Biosci. 317, 1749–1753 (2007).

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

Speidel, M.

Spudich, J. A.

L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
[Crossref]

Steinberg, R. A.

D. A. Agard, R. A. Steinberg, and R. M. Stroud, “Quantitative analysis of electrophoretograms: A mathematical approach to super-resolution,” Anal. Chem. 111, 257–268 (1981).

Straight, A.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Stroud, R. M.

D. A. Agard, R. A. Steinberg, and R. M. Stroud, “Quantitative analysis of electrophoretograms: A mathematical approach to super-resolution,” Anal. Chem. 111, 257–268 (1981).

Sturm, J.

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Tegenfeldt, J.

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Teleman, A.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Thomas, D. J.

J. M. Smith and D. J. Thomas, “Quantitative analysis of one-dimensional gel electrophoresis profiles,” Comput. Appl. Biosci. 317, 1749–1753 (2007).

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775–2783 (2002).
[Crossref] [PubMed]

Tomishige, M.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303, 676–678 (2004).
[Crossref]

Vale, R. D.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303, 676–678 (2004).
[Crossref]

Verkman, A. S.

H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position,” Biophys. J. 67, 1291–1300 (2007).
[Crossref]

Wang, Y. M.

M. C. DeSantis, S. H. DeCenzo, J. L. Li, and Y. M. Wang, “Precision analysis for standard deviation measurements of single-fluorescent molecule images,” Opt. Express 18, 6563–6576 (2010).
[Crossref] [PubMed]

Y. M. Wang, R. H. Austin, and E. C. Cox, “Single molecule measurements of repressor protein 1D diffusion on DNA,” Phys. Rev. Lett. 97, 048302 (2006).
[Crossref] [PubMed]

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

Ward, E. S.

S. Ram, E. S. Ward, and R. J. Ober, “Beyond Rayleigh’s criterion: A resolution measure with application to single-molecule microscopy,” Proc. Natl. Acad. Sci. USA 103, 4457–4462 (2006).
[Crossref] [PubMed]

Webb, C. D.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775–2783 (2002).
[Crossref] [PubMed]

Weiss, S.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

Wolf, E.

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, UK, 1999).

Wright, A.

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Wu, D.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).
[Crossref] [PubMed]

Yildiz, A.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303, 676–678 (2004).
[Crossref]

Zhuang, X.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

Anal. Chem. (1)

D. A. Agard, R. A. Steinberg, and R. M. Stroud, “Quantitative analysis of electrophoretograms: A mathematical approach to super-resolution,” Anal. Chem. 111, 257–268 (1981).

Appl. Phys. Lett. (1)

L Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90, 053902 (2007).
[Crossref]

Biophys. J. (2)

H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position,” Biophys. J. 67, 1291–1300 (2007).
[Crossref]

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775–2783 (2002).
[Crossref] [PubMed]

Cell (1)

G. S. Gordon, D. Sitnikov, C. D. Webb, A. Teleman, A. Straight, R. Losick, A. W. Murray, and A. Wright, “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechnisms,” Cell 90, 1113–1121 (1997).
[Crossref] [PubMed]

Comput. Appl. Biosci. (1)

J. M. Smith and D. J. Thomas, “Quantitative analysis of one-dimensional gel electrophoresis profiles,” Comput. Appl. Biosci. 317, 1749–1753 (2007).

Nanobiotechnology (1)

G. Han, P. Ghosh, M. De, and V. M. Rotello, “Drug and gene delivery using gold nanoparticles,” Nanobiotechnology 3, 40–45 (2007).
[Crossref]

Nature (1)

A. K. Salem, P. C. Searson, and K. W. Leong, “Multifunctional nanorods for gene delivery,” Nature 2, 668–671 (2003).
[Crossref]

Opt. Express (1)

Opt. Lett. (1)

Phys. Rev. Lett. (1)

Y. M. Wang, R. H. Austin, and E. C. Cox, “Single molecule measurements of repressor protein 1D diffusion on DNA,” Phys. Rev. Lett. 97, 048302 (2006).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. USA (6)

Y. M. Wang, J. Tegenfeldt, W. Reisner, R. Riehn, X.-J. Guan, L. Guo, I. Golding, E. C. Cox, J. Sturm, and R. H. Austin, “Single-molecule studies of repressor-DNA interactions show long-range interactions,” Proc. Natl. Acad. Sci. USA 102, 9796–9801 (2005).
[Crossref] [PubMed]

S. Ram, E. S. Ward, and R. J. Ober, “Beyond Rayleigh’s criterion: A resolution measure with application to single-molecule microscopy,” Proc. Natl. Acad. Sci. USA 103, 4457–4462 (2006).
[Crossref] [PubMed]

A. Sharonov and R. M. Hochstrasser, “Wide-field subdiffraction imaging by accumulated binding of diffusing probes,” Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).
[Crossref] [PubMed]

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multi-color colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA 97, 9461–9466 (2000).
[Crossref] [PubMed]

L. S. Churchman, Z. kten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. USA 105, 1419–1423 (2005).
[Crossref]

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).
[Crossref] [PubMed]

Science (3)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. L. Schwatz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303, 676–678 (2004).
[Crossref]

Single Molecules (1)

G. J. Schtz, V. Ph. Pastushenko, H. J. Gruber, H. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40 nm resolution,” Single Molecules 1, 25–31 (2000).
[Crossref]

Technometrics (1)

J. Behboodian, “On the modes of a mixture of two normal distributions,” Technometrics 12, 131–139 (1970).
[Crossref]

Other (1)

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, UK, 1999).

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Figures (5)

Fig. 1.
Fig. 1. Dimers of different subdiffraction separations. (A) From left to right, streptavidin-Cy3 dimers aligned in the horizontal direction separated by 0, 79, 158, and 237 nm. From the appearance, it is not clear that the image contains two molecules until near the 237 nm separation. The scale bar is 0.5 µm. (B) 1D intensity profiles of the dimers (circles are data and lines are the Gaussian fits) showing that the dimer SDs increase with separation.

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Fig. 2.
Fig. 2. Distinguishing dimers from monomers. (A) Experimental (gray) and simulated (empty bars) SD distributions of a constructed streptavidin-Cy3 monomer movie (left) and dimer movie (right) of separation 158 nm with the same photon count distribution of N = 2200 ± 156 photons (mean ± SD). The Gaussian fits to the experimental (solid lines) and simulated data (dashed lines) are shown. The simulations were for 1000 runs and the histograms were scaled to be comparable to the experimental data. (B) Comparing experimental (circles) and simulation (crosses) SD measurement errors Δsd of a 158 nm separated dimer at different detected photon counts. (C) Threshold photon count line (spline line) for distinguishing dimers from monomers at different separations. The points on the line are simulated data; circles are experimental data above the line, indicating differentiable dimers, and crosses are experimental data below the line, indicating monomers or non-differentiable dimers.

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Fig. 3.
Fig. 3. Dimer SD vs. separation diagram. The crosses are the mean SD values for simulations of 1000 dimer images, and the circles are experimental data at the separations of 0, 79, 158, and 237 nm. The error bars are the SDs of the SD distributions. A fit to the simulation results is the solid line. The horizontal grey lines off the extrema of the error bar of the 200 nm separation data meet the diagram, measuring the dimer separation measurement errors by the vertical grey lines.

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Fig. 4.
Fig. 4. Dimer separation measurement error, Δδ vs. separation at different photon counts of 150, 300, 400, 600, 2000, 10000, and 20,000 (slanted lines from light to dark). The low separation termination points of the lines are the separations below which dimers are not differentiable from monomers. The horizontal dashed lines are the dimer separation measurement error by using the centroid measurement method for dimer photon counts of 150 (top line) and 20,000 (bottom line).

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Fig. 5.
Fig. 5. SMID dimer separation measurement temporal resolution diagram. The line marks the threshold exposure time (threshold photon count) to differentiate dimers from monomers. The data points were converted from points in Fig. 2(C) using the conversion factor of 100 detected photons per 1 ms of exposure time, and the line is the splined connection of the data. The SMID timescales for all attainable subdiffraction dimer separation measurements are milliseconds.

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Equations (2)

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s d − s m > Δ s d + Δ s m .
f ( x , y ) = f 0 exp [ − ( x − x 0 ) 2 2 s x 2 − ( y − y 0 ) 2 2 s y 2 ] + 〈 b 〉 ,

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