Abstract

In microscopy, single fluorescence point sources can be localized with a precision several times greater than the resolution limit of the microscope. We show that the intermittent fluorescence or ‘blinking’ of quantum dots can analyzed by an Independent Component Analysis so as to identify the light emitted by each individual nanoparticle, localize it precisely, and thereby resolve groups of closely spaced (< λ/30) quantum dots. Both simulated and experimental data demonstrate that this technique is superior to localization based on Maximum Likelihood Estimation of the sum image under the assumption of point emitters. This technique has general application to any emitter with non-Gaussian temporal intensity distribution, including triplet state blinking. When applied to the labeling of structures, a high resolution “image” consisting of individually localized points may be reconstructed leading to the term “Pointillism”.

© 2005 Optical Society of America

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References

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  1. H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
    [Crossref]
  2. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
    [Crossref] [PubMed]
  3. D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
    [Crossref] [PubMed]
  4. M.G.L. Gustafsson, D.A. Agard, and J.W. Sedat. “I5M-3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195, 10–16 (1999).
    [Crossref] [PubMed]
  5. S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
    [Crossref]
  6. S.W. Hell and J. Wichmann. “Breaking the diffraction limit resolution by stimulated emission: stimulated-emission-depletion microscopy,” Opt. Lett. 19, 780–783 (1994).
    [Crossref] [PubMed]
  7. T.A. Klar, M. Dyba, and S.W. Hell. “Stimulated emission depletion microscopy with an offset depleting beam,” Appl. Phys. Lett. 78, 393–395 (2001).
    [Crossref]
  8. R. Heintzmann, T.M. Jovin, and C. Cremer. “Saturated patterned excitation microscopy - a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19, 1599–1609 (2002).
    [Crossref]
  9. R.J. Ober, S. Ram, and S.E. Ward. “Localization accuracy in single-molecule microscopy,” Biophys. J. 86, 1185–1200 (2004).
    [Crossref] [PubMed]
  10. A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
    [Crossref] [PubMed]
  11. A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
    [Crossref] [PubMed]
  12. M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
    [Crossref] [PubMed]
  13. M.P. Gordon, T. Ha, and P.R. Selvin. “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A. 101, 6462–6465 (2004).
    [Crossref] [PubMed]
  14. M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
    [Crossref]
  15. S. Hohng and T. Ha. “Near-complete suppression of quantum dot blinking in ambient conditions,” J. Am. Chem. Soc. 126, 1324–1325 (2004).
    [Crossref] [PubMed]
  16. M. Funaro, E. Oja, and H. Valpula. “Independent component analysis for artefact separation in astrophysical images,” Neural Networks 16, 469–478 (2003).
    [Crossref] [PubMed]
  17. J.-F. Cardoso. “Blind signal separation: statistical principles,” Proc. IEEE 86, 2009–2025 (1998).
    [Crossref]
  18. A. Hyvärinen. “Fast and robust fixed-point algorithms for independent component analysis,” IEEE Trans. Neural Networks 10, 626–634 (1999).
    [Crossref]
  19. J. Hurri, H. Gävert, J. Särelä, and A. Hyvärinen . FastICA: “An implementation of the fast fixed-point algorithm for independent component analysis in MATLAB, version 2.3, 2004,” Helsinki University of Technology, http://www.cis.hut.fi/projects/ica/fastica/.
  20. B. Richards and E. Wolf. “Electromagnetic diffraction in optical systems. II. structure of the image field in an aplanatic system,” Proc. R. Soc. London A 253, 358–379 (1959).
    [Crossref]
  21. T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
    [Crossref]
  22. R. Ando, H. Mizuno, and A. Miyawaki “Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting,” Science 306, 1370–1373 (2004).
    [Crossref] [PubMed]

2005 (2)

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
[Crossref]

2004 (6)

R. Ando, H. Mizuno, and A. Miyawaki “Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting,” Science 306, 1370–1373 (2004).
[Crossref] [PubMed]

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

R.J. Ober, S. Ram, and S.E. Ward. “Localization accuracy in single-molecule microscopy,” Biophys. J. 86, 1185–1200 (2004).
[Crossref] [PubMed]

M.P. Gordon, T. Ha, and P.R. Selvin. “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A. 101, 6462–6465 (2004).
[Crossref] [PubMed]

S. Hohng and T. Ha. “Near-complete suppression of quantum dot blinking in ambient conditions,” J. Am. Chem. Soc. 126, 1324–1325 (2004).
[Crossref] [PubMed]

2003 (2)

M. Funaro, E. Oja, and H. Valpula. “Independent component analysis for artefact separation in astrophysical images,” Neural Networks 16, 469–478 (2003).
[Crossref] [PubMed]

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

2002 (2)

R. Heintzmann, T.M. Jovin, and C. Cremer. “Saturated patterned excitation microscopy - a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19, 1599–1609 (2002).
[Crossref]

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

2001 (1)

T.A. Klar, M. Dyba, and S.W. Hell. “Stimulated emission depletion microscopy with an offset depleting beam,” Appl. Phys. Lett. 78, 393–395 (2001).
[Crossref]

2000 (1)

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

1999 (2)

M.G.L. Gustafsson, D.A. Agard, and J.W. Sedat. “I5M-3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[Crossref] [PubMed]

A. Hyvärinen. “Fast and robust fixed-point algorithms for independent component analysis,” IEEE Trans. Neural Networks 10, 626–634 (1999).
[Crossref]

1998 (1)

J.-F. Cardoso. “Blind signal separation: statistical principles,” Proc. IEEE 86, 2009–2025 (1998).
[Crossref]

1996 (1)

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

1994 (2)

S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[Crossref]

S.W. Hell and J. Wichmann. “Breaking the diffraction limit resolution by stimulated emission: stimulated-emission-depletion microscopy,” Opt. Lett. 19, 780–783 (1994).
[Crossref] [PubMed]

1959 (1)

B. Richards and E. Wolf. “Electromagnetic diffraction in optical systems. II. structure of the image field in an aplanatic system,” Proc. R. Soc. London A 253, 358–379 (1959).
[Crossref]

Agard, D.A.

M.G.L. Gustafsson, D.A. Agard, and J.W. Sedat. “I5M-3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[Crossref] [PubMed]

Amariglio, N.

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

Ando, R.

R. Ando, H. Mizuno, and A. Miyawaki “Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting,” Science 306, 1370–1373 (2004).
[Crossref] [PubMed]

Arndt-Jovin, D.J.

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

Bawendi, M.G.

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Bentolila, L. A.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Bezuijen, M.

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
[Crossref]

Brus, L.E.

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Cardoso, J.-F.

J.-F. Cardoso. “Blind signal separation: statistical principles,” Proc. IEEE 86, 2009–2025 (1998).
[Crossref]

Cremer, C.

R. Heintzmann, T.M. Jovin, and C. Cremer. “Saturated patterned excitation microscopy - a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19, 1599–1609 (2002).
[Crossref]

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[Crossref]

Dabbausi, B.O.

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Doose, S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Dyba, M.

T.A. Klar, M. Dyba, and S.W. Hell. “Stimulated emission depletion microscopy with an offset depleting beam,” Appl. Phys. Lett. 78, 393–395 (2001).
[Crossref]

Edelmann, P.

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

Esa, A.

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

Forkey, J.N.

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

Funaro, M.

M. Funaro, E. Oja, and H. Valpula. “Independent component analysis for artefact separation in astrophysical images,” Neural Networks 16, 469–478 (2003).
[Crossref] [PubMed]

Gambhir, S. S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Gävert, H.

J. Hurri, H. Gävert, J. Särelä, and A. Hyvärinen . FastICA: “An implementation of the fast fixed-point algorithm for independent component analysis in MATLAB, version 2.3, 2004,” Helsinki University of Technology, http://www.cis.hut.fi/projects/ica/fastica/.

Goldman, Y.E.

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

Gordon, M.P.

M.P. Gordon, T. Ha, and P.R. Selvin. “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A. 101, 6462–6465 (2004).
[Crossref] [PubMed]

Grecco, H.

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

Gustafsson, M.G.L.

M.G.L. Gustafsson, D.A. Agard, and J.W. Sedat. “I5M-3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[Crossref] [PubMed]

Ha, T.

M.P. Gordon, T. Ha, and P.R. Selvin. “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A. 101, 6462–6465 (2004).
[Crossref] [PubMed]

S. Hohng and T. Ha. “Near-complete suppression of quantum dot blinking in ambient conditions,” J. Am. Chem. Soc. 126, 1324–1325 (2004).
[Crossref] [PubMed]

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

Harris, T.D.

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Hausmann, M.

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

Heilemann, M.

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Heintzmann, R.

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

R. Heintzmann, T.M. Jovin, and C. Cremer. “Saturated patterned excitation microscopy - a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19, 1599–1609 (2002).
[Crossref]

Hell, S.W.

T.A. Klar, M. Dyba, and S.W. Hell. “Stimulated emission depletion microscopy with an offset depleting beam,” Appl. Phys. Lett. 78, 393–395 (2001).
[Crossref]

S.W. Hell and J. Wichmann. “Breaking the diffraction limit resolution by stimulated emission: stimulated-emission-depletion microscopy,” Opt. Lett. 19, 780–783 (1994).
[Crossref] [PubMed]

S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[Crossref]

Herten, D.P.

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Hohng, S.

S. Hohng and T. Ha. “Near-complete suppression of quantum dot blinking in ambient conditions,” J. Am. Chem. Soc. 126, 1324–1325 (2004).
[Crossref] [PubMed]

Hurri, J.

J. Hurri, H. Gävert, J. Särelä, and A. Hyvärinen . FastICA: “An implementation of the fast fixed-point algorithm for independent component analysis in MATLAB, version 2.3, 2004,” Helsinki University of Technology, http://www.cis.hut.fi/projects/ica/fastica/.

Hyvärinen, A.

A. Hyvärinen. “Fast and robust fixed-point algorithms for independent component analysis,” IEEE Trans. Neural Networks 10, 626–634 (1999).
[Crossref]

J. Hurri, H. Gävert, J. Särelä, and A. Hyvärinen . FastICA: “An implementation of the fast fixed-point algorithm for independent component analysis in MATLAB, version 2.3, 2004,” Helsinki University of Technology, http://www.cis.hut.fi/projects/ica/fastica/.

Jares-Erijman, E.A.

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

Jovin, T.M.

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

R. Heintzmann, T.M. Jovin, and C. Cremer. “Saturated patterned excitation microscopy - a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19, 1599–1609 (2002).
[Crossref]

Klar, T.A.

T.A. Klar, M. Dyba, and S.W. Hell. “Stimulated emission depletion microscopy with an offset depleting beam,” Appl. Phys. Lett. 78, 393–395 (2001).
[Crossref]

Kreth, G.

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

Li, J. J.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Lidke, D.S.

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

Lidke, K.A.

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

Lindek, S.

S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[Crossref]

Luengo Hendriks, C.L.

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
[Crossref]

Macklin, J.J.

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Martinez, O.E.

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

McKinney, S.A.

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

Michalet, X.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Miyawaki, A.

R. Ando, H. Mizuno, and A. Miyawaki “Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting,” Science 306, 1370–1373 (2004).
[Crossref] [PubMed]

Mizuno, H.

R. Ando, H. Mizuno, and A. Miyawaki “Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting,” Science 306, 1370–1373 (2004).
[Crossref] [PubMed]

Müller, C.

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Nagy, P.

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

Nirmal, M.

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Ober, R.J.

R.J. Ober, S. Ram, and S.E. Ward. “Localization accuracy in single-molecule microscopy,” Biophys. J. 86, 1185–1200 (2004).
[Crossref] [PubMed]

Oja, E.

M. Funaro, E. Oja, and H. Valpula. “Independent component analysis for artefact separation in astrophysical images,” Neural Networks 16, 469–478 (2003).
[Crossref] [PubMed]

Pham, T.Q.

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
[Crossref]

Pinaud, F. F.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Post, J.N.

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

Ram, S.

R.J. Ober, S. Ram, and S.E. Ward. “Localization accuracy in single-molecule microscopy,” Biophys. J. 86, 1185–1200 (2004).
[Crossref] [PubMed]

Rechavi, G.

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

Richards, B.

B. Richards and E. Wolf. “Electromagnetic diffraction in optical systems. II. structure of the image field in an aplanatic system,” Proc. R. Soc. London A 253, 358–379 (1959).
[Crossref]

Särelä, J.

J. Hurri, H. Gävert, J. Särelä, and A. Hyvärinen . FastICA: “An implementation of the fast fixed-point algorithm for independent component analysis in MATLAB, version 2.3, 2004,” Helsinki University of Technology, http://www.cis.hut.fi/projects/ica/fastica/.

Sauer, M.

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Schutte, K.

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
[Crossref]

Sedat, J.W.

M.G.L. Gustafsson, D.A. Agard, and J.W. Sedat. “I5M-3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[Crossref] [PubMed]

Selvin, P.R.

M.P. Gordon, T. Ha, and P.R. Selvin. “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A. 101, 6462–6465 (2004).
[Crossref] [PubMed]

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

Spagnuolo, C.

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

Stelzer, E.H.K.

S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[Crossref]

Sundaresan, G.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Tinnefeld, P.

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Trakhtenbrot, L.

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

Trautman, J.K.

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Tsay, J. M.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Valpula, H.

M. Funaro, E. Oja, and H. Valpula. “Independent component analysis for artefact separation in astrophysical images,” Neural Networks 16, 469–478 (2003).
[Crossref] [PubMed]

van Vliet, L.J.

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
[Crossref]

Ward, S.E.

R.J. Ober, S. Ram, and S.E. Ward. “Localization accuracy in single-molecule microscopy,” Biophys. J. 86, 1185–1200 (2004).
[Crossref] [PubMed]

Weiss, S.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Weston, K.D.

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Wichmann, J.

Wolf, E.

B. Richards and E. Wolf. “Electromagnetic diffraction in optical systems. II. structure of the image field in an aplanatic system,” Proc. R. Soc. London A 253, 358–379 (1959).
[Crossref]

Wolfrum, J.

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Wu, A. M.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Yildiz, A.

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

Anal. Chem. (1)

M. Heilemann, D.P. Herten, R. Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum, and M. Sauer. “High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy,” Anal. Chem. 74, 3511–3517 (2002).
[Crossref] [PubMed]

Appl. Phys. Lett. (2)

S.W. Hell, S. Lindek, C. Cremer, and E.H.K. Stelzer. “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994).
[Crossref]

T.A. Klar, M. Dyba, and S.W. Hell. “Stimulated emission depletion microscopy with an offset depleting beam,” Appl. Phys. Lett. 78, 393–395 (2001).
[Crossref]

Biophys. J. (1)

R.J. Ober, S. Ram, and S.E. Ward. “Localization accuracy in single-molecule microscopy,” Biophys. J. 86, 1185–1200 (2004).
[Crossref] [PubMed]

IEEE Trans. Neural Networks (1)

A. Hyvärinen. “Fast and robust fixed-point algorithms for independent component analysis,” IEEE Trans. Neural Networks 10, 626–634 (1999).
[Crossref]

J. Am. Chem. Soc. (1)

S. Hohng and T. Ha. “Near-complete suppression of quantum dot blinking in ambient conditions,” J. Am. Chem. Soc. 126, 1324–1325 (2004).
[Crossref] [PubMed]

J. Microsc. (2)

A. Esa, P. Edelmann, G. Kreth, L. Trakhtenbrot, N. Amariglio, G. Rechavi, M. Hausmann, and C. Cremer. “Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000).
[Crossref] [PubMed]

M.G.L. Gustafsson, D.A. Agard, and J.W. Sedat. “I5M-3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195, 10–16 (1999).
[Crossref] [PubMed]

J. Opt. Soc. Am. A (1)

Microsc. Res. Tech. (1)

H. Grecco, K.A. Lidke, R. Heintzmann, D.S. Lidke, C. Spagnuolo, O.E. Martinez, E.A. Jares-Erijman, and T.M. Jovin. “Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells,” Microsc. Res. Tech. 65, 169–179 (2004).
[Crossref]

Nat. Biotechnol. (1)

D.S. Lidke, P. Nagy, R. Heintzmann, D.J. Arndt-Jovin, J.N. Post, H. Grecco, E.A. Jares-Erijman, and T.M. Jovin. “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nat. Biotechnol. 22, 198–203 (2004).
[Crossref] [PubMed]

Nature (1)

M. Nirmal, B.O. Dabbausi, M.G. Bawendi, J.J. Macklin, J.K. Trautman, T.D. Harris, and L.E. Brus. “Fluoroscence intermittency in single cadmium selenide nanocrystals,” Nature 383, 802–805 (1996).
[Crossref]

Neural Networks (1)

M. Funaro, E. Oja, and H. Valpula. “Independent component analysis for artefact separation in astrophysical images,” Neural Networks 16, 469–478 (2003).
[Crossref] [PubMed]

Opt. Lett. (1)

Proc. IEEE (1)

J.-F. Cardoso. “Blind signal separation: statistical principles,” Proc. IEEE 86, 2009–2025 (1998).
[Crossref]

Proc. Natl. Acad. Sci. U.S.A. (1)

M.P. Gordon, T. Ha, and P.R. Selvin. “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A. 101, 6462–6465 (2004).
[Crossref] [PubMed]

Proc. R. Soc. London A (1)

B. Richards and E. Wolf. “Electromagnetic diffraction in optical systems. II. structure of the image field in an aplanatic system,” Proc. R. Soc. London A 253, 358–379 (1959).
[Crossref]

Proc. SPIE (1)

T.Q. Pham, M. Bezuijen, L.J. van Vliet, K. Schutte, and C.L. Luengo Hendriks. “Performance of optimal registration estimators,” Proc. SPIE 5817, 133–144 (2005).
[Crossref]

Science (3)

R. Ando, H. Mizuno, and A. Miyawaki “Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting,” Science 306, 1370–1373 (2004).
[Crossref] [PubMed]

A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin. “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5 nm localization,” Science 300, 2061–2066 (2003).
[Crossref] [PubMed]

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005).
[Crossref] [PubMed]

Other (1)

J. Hurri, H. Gävert, J. Särelä, and A. Hyvärinen . FastICA: “An implementation of the fast fixed-point algorithm for independent component analysis in MATLAB, version 2.3, 2004,” Helsinki University of Technology, http://www.cis.hut.fi/projects/ica/fastica/.

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Figures (3)

Fig. 1.
Fig. 1.

Ability of the ICA approach to localize two blinking sources compared to fitting the sum image to a 2 source model with a maximum likelihood estimate. Each point represents the average of 50 simulations. To account for outliers (found only in the MLE analysis), localization errors > 4 pixels were ignored. The photon counts refer to the expected maximum photons per dot per image. Each pixel corresponded to 108 nm as given by the geometry of the experiments. The black line indicates where the localization error was equal to the separation. (a) The mean localization error divided by separation (b) 1000 expected max photons as for one of the above cases but with the data series and the PSF smoothed (Gaussian kernel with indicated width σ) in x and y before analysis. A smoothing with σ = 1 pixel leads to a beneficial trade off between signal to noise and spatial resolution.

Fig. 2.
Fig. 2.

The results of the ICA decomposition for three examples of experimentally acquired blinking QD data sets. Top: 10 frames from the time series. Bottom, left to right: Sum image of time series, ICA returned component 1, ICA returned component 2, color overlay of component 1 and 2. Images are 32×32 pixels with pixel size of 108 nm. (a) Two widely separated blinking quantum dots, separation distance is found to be 435 nm. (b) Two closely spaced QDs, separation is found to be 23 nm. (c) Only a single quantum dot is found, the other component is noise.

Fig. 3.
Fig. 3.

The results of the ICA decomposition of 3 blinking emitters. The coordinates in pixels with respect to the center of the image are (0.9501,-0.2311), (1.3380,-0.5467) and (0.9881,-0.7297). The data series was simulated with 1000 photons per dot per QD and 500 time frames. Images are 16×16 pixels with a pixel size of 108 nm. (a and b) are the images found when the ICA analysis assumes 2 emitters. Localization yields coordinates of (0.9801,-0.2139) and (1.1497,-0.6448). (c, d, and e) are the images found when the ICA analysis assumes 3 emitters. Localization yields coordinates (1.0024,-0.7596),(1.3447,-0.5623) and (0.9553,-0.2648). (d, e, f and g) are the images found when the ICA analysis assumes 4 emitters. Localization yields coordinates of (1.3397 -0.5558),(0.9487 -0.2636),(0.0485 0.2060), and (0.9897 -0.7659).

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