Abstract

Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross- and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 ± 12 nm diameter, and 40 nm domains were estimated to 65 ± 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.

© 2014 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. S. C. Prinster, C. Hague, R. A. Hall, “Heterodimerization of g protein-coupled receptors: specificity and functional significance,” Pharmacol. Rev. 57(3), 289–298 (2005).
    [CrossRef] [PubMed]
  2. I. Bethani, S. S. Skånland, I. Dikic, A. Acker-Palmer, “Spatial organization of transmembrane receptor signalling,” EMBO J. 29(16), 2677–2688 (2010).
    [CrossRef] [PubMed]
  3. D. Lingwood, K. Simons, “Lipid Rafts As a Membrane-Organizing Principle,” Science 327(5961), 46–50 (2010).
    [CrossRef] [PubMed]
  4. S. W. Hell, J. Wichmann, “Breaking the Diffraction Resolution Limit by Stimulated Emission: Stimulated-Emission-Depletion Fluorescence Microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [CrossRef] [PubMed]
  5. M. J. Rust, M. Bates, X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
    [CrossRef] [PubMed]
  6. M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
    [CrossRef] [PubMed]
  7. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
    [CrossRef] [PubMed]
  8. V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
    [CrossRef] [PubMed]
  9. C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
    [CrossRef] [PubMed]
  10. D. Magde, W. W. Webb, E. Elson, “Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
    [CrossRef]
  11. R. Rigler, U. Mets, J. Widengren, P. Kask, “Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,” Eur Biophys J Biophy 22(3), 169–175 (1993).
    [CrossRef]
  12. J. Widengren, R. Rigler, “Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces,” Cell. Mol. Biol. (Noisy-le-grand) 44(5), 857–879 (1998).
    [PubMed]
  13. S. Wennmalm, P. Thyberg, L. Xu, J. Widengren, “Inverse-fluorescence correlation spectroscopy,” Anal. Chem. 81(22), 9209–9215 (2009).
    [CrossRef] [PubMed]
  14. S. Wennmalm, J. Widengren, “Inverse-fluorescence cross-correlation spectroscopy,” Anal. Chem. 82(13), 5646–5651 (2010).
    [CrossRef] [PubMed]
  15. S. Wennmalm, J. Widengren, “Inverse-fluorescence correlation spectroscopy: more information and less labeling,” Front Biosci (Schol Ed) 3(1), 385–392 (2011).
    [CrossRef] [PubMed]
  16. E. Elson, Washington University, St Louis, Missouri, USA, (personal communication, 2013).
  17. T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
    [CrossRef] [PubMed]
  18. D. Wildanger, E. Rittweger, L. Kastrup, S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
    [CrossRef] [PubMed]
  19. D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
    [CrossRef] [PubMed]
  20. S. Costantino, J. W. D. Comeau, D. L. Kolin, P. W. Wiseman, “Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy,” Biophys. J. 89(2), 1251–1260 (2005).
    [CrossRef] [PubMed]
  21. N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
    [CrossRef] [PubMed]
  22. D. L. Kolin, P. W. Wiseman, “Advances in image correlation spectroscopy: Measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
    [CrossRef] [PubMed]
  23. A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
    [CrossRef] [PubMed]
  24. F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
    [CrossRef] [PubMed]
  25. B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
    [CrossRef] [PubMed]

2013 (2)

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

2012 (2)

D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
[CrossRef] [PubMed]

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

2011 (1)

S. Wennmalm, J. Widengren, “Inverse-fluorescence correlation spectroscopy: more information and less labeling,” Front Biosci (Schol Ed) 3(1), 385–392 (2011).
[CrossRef] [PubMed]

2010 (4)

S. Wennmalm, J. Widengren, “Inverse-fluorescence cross-correlation spectroscopy,” Anal. Chem. 82(13), 5646–5651 (2010).
[CrossRef] [PubMed]

I. Bethani, S. S. Skånland, I. Dikic, A. Acker-Palmer, “Spatial organization of transmembrane receptor signalling,” EMBO J. 29(16), 2677–2688 (2010).
[CrossRef] [PubMed]

D. Lingwood, K. Simons, “Lipid Rafts As a Membrane-Organizing Principle,” Science 327(5961), 46–50 (2010).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

2009 (2)

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

S. Wennmalm, P. Thyberg, L. Xu, J. Widengren, “Inverse-fluorescence correlation spectroscopy,” Anal. Chem. 81(22), 9209–9215 (2009).
[CrossRef] [PubMed]

2008 (3)

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

2007 (1)

D. L. Kolin, P. W. Wiseman, “Advances in image correlation spectroscopy: Measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

2006 (2)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

2005 (2)

S. C. Prinster, C. Hague, R. A. Hall, “Heterodimerization of g protein-coupled receptors: specificity and functional significance,” Pharmacol. Rev. 57(3), 289–298 (2005).
[CrossRef] [PubMed]

S. Costantino, J. W. D. Comeau, D. L. Kolin, P. W. Wiseman, “Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy,” Biophys. J. 89(2), 1251–1260 (2005).
[CrossRef] [PubMed]

1998 (1)

J. Widengren, R. Rigler, “Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces,” Cell. Mol. Biol. (Noisy-le-grand) 44(5), 857–879 (1998).
[PubMed]

1994 (1)

1993 (2)

R. Rigler, U. Mets, J. Widengren, P. Kask, “Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,” Eur Biophys J Biophy 22(3), 169–175 (1993).
[CrossRef]

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

1972 (1)

D. Magde, W. W. Webb, E. Elson, “Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Acker-Palmer, A.

I. Bethani, S. S. Skånland, I. Dikic, A. Acker-Palmer, “Spatial organization of transmembrane receptor signalling,” EMBO J. 29(16), 2677–2688 (2010).
[CrossRef] [PubMed]

Auer, G.

D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
[CrossRef] [PubMed]

Bates, M.

M. J. Rust, M. Bates, X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Belov, V. N.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Berning, S.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

Bethani, I.

I. Bethani, S. S. Skånland, I. Dikic, A. Acker-Palmer, “Spatial organization of transmembrane receptor signalling,” EMBO J. 29(16), 2677–2688 (2010).
[CrossRef] [PubMed]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Blom, H.

D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
[CrossRef] [PubMed]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Comeau, J. W. D.

S. Costantino, J. W. D. Comeau, D. L. Kolin, P. W. Wiseman, “Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy,” Biophys. J. 89(2), 1251–1260 (2005).
[CrossRef] [PubMed]

Cordes, V. C.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

Costantino, S.

S. Costantino, J. W. D. Comeau, D. L. Kolin, P. W. Wiseman, “Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy,” Biophys. J. 89(2), 1251–1260 (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. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Deluz, C.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

Diederichsen, U.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Dikic, I.

I. Bethani, S. S. Skånland, I. Dikic, A. Acker-Palmer, “Spatial organization of transmembrane receptor signalling,” EMBO J. 29(16), 2677–2688 (2010).
[CrossRef] [PubMed]

Eggeling, C.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Elson, E.

D. Magde, W. W. Webb, E. Elson, “Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Fasshauer, D.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Göttfert, F.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

Grubmüller, H.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Hague, C.

S. C. Prinster, C. Hague, R. A. Hall, “Heterodimerization of g protein-coupled receptors: specificity and functional significance,” Pharmacol. Rev. 57(3), 289–298 (2005).
[CrossRef] [PubMed]

Hall, R. A.

S. C. Prinster, C. Hague, R. A. Hall, “Heterodimerization of g protein-coupled receptors: specificity and functional significance,” Pharmacol. Rev. 57(3), 289–298 (2005).
[CrossRef] [PubMed]

Hassaïne, G.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

Heilemann, M.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

Hein, B.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Hell, S. W.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

S. W. Hell, J. Wichmann, “Breaking the Diffraction Resolution Limit by Stimulated Emission: Stimulated-Emission-Depletion Fluorescence Microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[CrossRef] [PubMed]

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Höddelius, P. L.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Honigmann, A.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

Iraheta, E.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Jahn, R.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Jakobs, S.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Kamin, D.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Kask, P.

R. Rigler, U. Mets, J. Widengren, P. Kask, “Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,” Eur Biophys J Biophy 22(3), 169–175 (1993).
[CrossRef]

Kasper, R.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

Kastrup, L.

Kolin, D. L.

D. L. Kolin, P. W. Wiseman, “Advances in image correlation spectroscopy: Measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

S. Costantino, J. W. D. Comeau, D. L. Kolin, P. W. Wiseman, “Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy,” Biophys. J. 89(2), 1251–1260 (2005).
[CrossRef] [PubMed]

Kühnel, K.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Lauterbach, M. A.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lingwood, D.

D. Lingwood, K. Simons, “Lipid Rafts As a Membrane-Organizing Principle,” Science 327(5961), 46–50 (2010).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Magde, D.

D. Magde, W. W. Webb, E. Elson, “Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Magnusson, K. E.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Martin, O. J.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

Medda, R.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Mets, U.

R. Rigler, U. Mets, J. Widengren, P. Kask, “Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,” Eur Biophys J Biophy 22(3), 169–175 (1993).
[CrossRef]

Milovanovic, D.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Mueller, V.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

Mukherjee, A.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

Müllar, S.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Petersen, N. O.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Polyakova, S.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Prinster, S. C.

S. C. Prinster, C. Hague, R. A. Hall, “Heterodimerization of g protein-coupled receptors: specificity and functional significance,” Pharmacol. Rev. 57(3), 289–298 (2005).
[CrossRef] [PubMed]

Rigler, R.

J. Widengren, R. Rigler, “Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces,” Cell. Mol. Biol. (Noisy-le-grand) 44(5), 857–879 (1998).
[PubMed]

R. Rigler, U. Mets, J. Widengren, P. Kask, “Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,” Eur Biophys J Biophy 22(3), 169–175 (1993).
[CrossRef]

Ringemann, C.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Risselada, H. J.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Rittweger, E.

Rizzoli, S. O.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Rönnlund, D.

D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
[CrossRef] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Sandén, T.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

Sandhoff, K.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Santschi, C.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

Sauer, M.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

Schönle, A.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Schüttpelz, M.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

Schwarzmann, G.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Seefeldt, B.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

Seger, O.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Simons, K.

D. Lingwood, K. Simons, “Lipid Rafts As a Membrane-Organizing Principle,” Science 327(5961), 46–50 (2010).
[CrossRef] [PubMed]

Skånland, S. S.

I. Bethani, S. S. Skånland, I. Dikic, A. Acker-Palmer, “Spatial organization of transmembrane receptor signalling,” EMBO J. 29(16), 2677–2688 (2010).
[CrossRef] [PubMed]

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Thyberg, P.

S. Wennmalm, P. Thyberg, L. Xu, J. Widengren, “Inverse-fluorescence correlation spectroscopy,” Anal. Chem. 81(22), 9209–9215 (2009).
[CrossRef] [PubMed]

Tinnefeld, P.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

van de Linde, S.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

van den Bogaart, G.

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Vogel, H.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

von Middendorff, C.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Webb, W. W.

D. Magde, W. W. Webb, E. Elson, “Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Wennmalm, S.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

S. Wennmalm, J. Widengren, “Inverse-fluorescence correlation spectroscopy: more information and less labeling,” Front Biosci (Schol Ed) 3(1), 385–392 (2011).
[CrossRef] [PubMed]

S. Wennmalm, J. Widengren, “Inverse-fluorescence cross-correlation spectroscopy,” Anal. Chem. 82(13), 5646–5651 (2010).
[CrossRef] [PubMed]

S. Wennmalm, P. Thyberg, L. Xu, J. Widengren, “Inverse-fluorescence correlation spectroscopy,” Anal. Chem. 81(22), 9209–9215 (2009).
[CrossRef] [PubMed]

Westphal, V.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Wichmann, J.

Widengren, J.

D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
[CrossRef] [PubMed]

S. Wennmalm, J. Widengren, “Inverse-fluorescence correlation spectroscopy: more information and less labeling,” Front Biosci (Schol Ed) 3(1), 385–392 (2011).
[CrossRef] [PubMed]

S. Wennmalm, J. Widengren, “Inverse-fluorescence cross-correlation spectroscopy,” Anal. Chem. 82(13), 5646–5651 (2010).
[CrossRef] [PubMed]

S. Wennmalm, P. Thyberg, L. Xu, J. Widengren, “Inverse-fluorescence correlation spectroscopy,” Anal. Chem. 81(22), 9209–9215 (2009).
[CrossRef] [PubMed]

J. Widengren, R. Rigler, “Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces,” Cell. Mol. Biol. (Noisy-le-grand) 44(5), 857–879 (1998).
[PubMed]

R. Rigler, U. Mets, J. Widengren, P. Kask, “Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,” Eur Biophys J Biophy 22(3), 169–175 (1993).
[CrossRef]

Wildanger, D.

Willig, K. I.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Wiseman, P. W.

D. L. Kolin, P. W. Wiseman, “Advances in image correlation spectroscopy: Measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

S. Costantino, J. W. D. Comeau, D. L. Kolin, P. W. Wiseman, “Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy,” Biophys. J. 89(2), 1251–1260 (2005).
[CrossRef] [PubMed]

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Wurm, C. A.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Wyss, R.

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

Xu, L.

S. Wennmalm, P. Thyberg, L. Xu, J. Widengren, “Inverse-fluorescence correlation spectroscopy,” Anal. Chem. 81(22), 9209–9215 (2009).
[CrossRef] [PubMed]

Yang, Y.

D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
[CrossRef] [PubMed]

Zhuang, X. W.

M. J. Rust, M. Bates, X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Adv Healthc Mater (1)

D. Rönnlund, Y. Yang, H. Blom, G. Auer, J. Widengren, “Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications,” Adv Healthc Mater 1(6), 707–713 (2012).
[CrossRef] [PubMed]

Anal. Chem. (2)

S. Wennmalm, P. Thyberg, L. Xu, J. Widengren, “Inverse-fluorescence correlation spectroscopy,” Anal. Chem. 81(22), 9209–9215 (2009).
[CrossRef] [PubMed]

S. Wennmalm, J. Widengren, “Inverse-fluorescence cross-correlation spectroscopy,” Anal. Chem. 82(13), 5646–5651 (2010).
[CrossRef] [PubMed]

Angew. Chem. Int. Ed. Engl. (1)

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[CrossRef] [PubMed]

Biophys. J. (4)

S. Costantino, J. W. D. Comeau, D. L. Kolin, P. W. Wiseman, “Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy,” Biophys. J. 89(2), 1251–1260 (2005).
[CrossRef] [PubMed]

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, K. E. Magnusson, “Quantitation of Membrane Receptor Distributions by Image Correlation Spectroscopy: Concept and Application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, S. W. Hell, “Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Cell Biochem. Biophys. (1)

D. L. Kolin, P. W. Wiseman, “Advances in image correlation spectroscopy: Measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

Cell. Mol. Biol. (Noisy-le-grand) (1)

J. Widengren, R. Rigler, “Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces,” Cell. Mol. Biol. (Noisy-le-grand) 44(5), 857–879 (1998).
[PubMed]

EMBO J. (1)

I. Bethani, S. S. Skånland, I. Dikic, A. Acker-Palmer, “Spatial organization of transmembrane receptor signalling,” EMBO J. 29(16), 2677–2688 (2010).
[CrossRef] [PubMed]

Eur Biophys J Biophy (1)

R. Rigler, U. Mets, J. Widengren, P. Kask, “Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,” Eur Biophys J Biophy 22(3), 169–175 (1993).
[CrossRef]

Front Biosci (Schol Ed) (1)

S. Wennmalm, J. Widengren, “Inverse-fluorescence correlation spectroscopy: more information and less labeling,” Front Biosci (Schol Ed) 3(1), 385–392 (2011).
[CrossRef] [PubMed]

Nano Lett. (1)

T. Sandén, R. Wyss, C. Santschi, G. Hassaïne, C. Deluz, O. J. Martin, S. Wennmalm, H. Vogel, “A zeptoliter volume meter for analysis of single protein molecules,” Nano Lett. 12(1), 370–375 (2012).
[CrossRef] [PubMed]

Nat. Methods (1)

M. J. Rust, M. Bates, X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Nat. Struct. Mol. Biol. (1)

A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, “Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment,” Nat. Struct. Mol. Biol. 20(6), 679–686 (2013).
[CrossRef] [PubMed]

Nature (1)

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (1)

Pharmacol. Rev. (1)

S. C. Prinster, C. Hague, R. A. Hall, “Heterodimerization of g protein-coupled receptors: specificity and functional significance,” Pharmacol. Rev. 57(3), 289–298 (2005).
[CrossRef] [PubMed]

Phys. Rev. Lett. (1)

D. Magde, W. W. Webb, E. Elson, “Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Science (3)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

D. Lingwood, K. Simons, “Lipid Rafts As a Membrane-Organizing Principle,” Science 327(5961), 46–50 (2010).
[CrossRef] [PubMed]

Other (1)

E. Elson, Washington University, St Louis, Missouri, USA, (personal communication, 2013).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (12)

Fig. 1
Fig. 1

A typical image of red NPs (A) and the corresponding cross-talk image (B). The white square marks an area where the average intensity is compared between the images. For this particular area the mean intensity in the original image (A) was Ir 5 counts and the mean intensity in the cross-talk image (B) was ICT 15 counts giving F ≈ 0.3.

Fig. 2
Fig. 2

Simulated images, including noise, with 100 and 1000 red and green NPs respectively. (A) Simulated confocal image of size 100 × 100 pixels, NP size was 1 pixel and the resolution was 6 pixels. This corresponds roughly to confocal imaging of 40 nm NPs (compare with Fig. 9(A)). (B) Simulated STED image. The location of the NPs is not the same as in Fig. 2(A). Image size was 250 × 250 pixels, NP size was 1 pixel and the resolution was 4 pixels. This corresponds roughly to STED imaging of the 40 nm NPs (compare with Fig. 9(B)).

Fig. 3
Fig. 3

Cross-correlation curves for simulated images containing 100 × 100 pixels. The NP size was 1 pixel and the number of red and green NPs was 100 and 1000 respectively. The resolution was 6 pixels. (A) Examples of single-image cross-correlation curves. A curve with positive amplitude (blue) and a curve with negative amplitude (red) are shown. (B) Average correlation curves for 100 images: of all cross-correlation curves (blue), and of all cross-correlation curves with negative amplitude, 53 in total.

Fig. 4
Fig. 4

The intensity profiles of two green NPs surrounding a red NP. The yellow areas indicate the overlap of the intensity profiles which may contribute positively to the cross-correlation amplitude. (A) Small overlap of the intensity profiles. (B) Large overlap of the intensity profiles.

Fig. 5
Fig. 5

Typical confocal (A) and STED image (B) of the same scanned area on the cover slip. The size of the scanned area was 5 × 5 μm. The white arrow points out an area on the surface were the NPs most likely had formed a multiple layer, which however had little effect on the analysis. (C) Trace for arbitrary line in the confocal image.

Fig. 6
Fig. 6

Typical cross correlation curve (A) and auto-correlation (B) curves for a single image of the 200 nm NPs. On the x-axis 1 pixel corresponds to 50 nm.

Fig. 7
Fig. 7

Average cross- (A) and auto correlation (B) curves for all 14 raw images of the 250 nm NPs. The Gaussian fit of the data points gave the amplitudes GCC(0) = −0.22 and GAC,r(0) = 1.95. The decay width at e−2 was 267 nm for both curves. On the x-axis 1 pixel corresponds to 50 nm.

Fig. 8
Fig. 8

Average cross- (A) and auto-correlation (B) curves for all 14 cross-talk reduced images of the 250 nm NPs. The amplitudes were GCC(0) = −0.27 and GAC,r(0) = 2.15 and the decay widths at e−2 was 270 nm for both curves. On the x-axis 1 pixel corresponds to 50 nm.

Fig. 9
Fig. 9

Typical confocal (A) and STED (B) images of the same 5 × 5 μm area of 40 nm NPs. In the confocal image the individual NPs cannot be resolved because of the resolution of ~270 nm (compare with Fig. 5(A)). For the STED image the ~40 nm resolution can almost distinguish individual NPs.

Fig. 10
Fig. 10

(A) and (B): Average cross- and auto-correlation curves for all raw confocal images yielding anti-correlation, 19 out of 42, of the 40 nm NPs. The amplitudes were GCC(0) = −0.08 and GAC,r(0) = 0.46. The decay widths at e−2 were 410 nm 290 nm for the cross- and the auto-correlation curves respectively. On the x-axis 1 pixel corresponds to 50 nm. (C) and (D): Average curves from the 19 out of 42 STED images that yielded anti-correlation. The amplitudes were GCC(0) = −0.013 and GAC,r(0) = 1.98. The decay widths at e−2 were 156 nm and 170 nm for the cross- and the auto-correlation curves respectively. On the x-axis 1 pixel corresponds to 20 nm.

Fig. 11
Fig. 11

Average cross-correlation curve of the cross-talk compensated confocal images of 40 nm NPs yielding negative amplitude (A) and the corresponding auto-correlation curve (B). For comparison the average cross-correlation curves from confocal images of the 40 nm NPs and the 250 nm NPs are displayed in the same graph (C). On the x-axis 1 pixel corresponds to 50 nm.

Fig. 12
Fig. 12

Average cross-correlation curve of the cross-talk compensated STED images yielding negative amplitude, 38 out of 42 (A), and the corresponding auto-correlation curve (B). The amplitudes were GCC(0) = −0.12 and GAC,r(0) = 6.46. The decay widths at e−2 were 156 nm and 154 nm for the cross- and the auto-correlation curves respectively. On the x-axis 1 pixel corresponds to 20 nm.

Tables (4)

Tables Icon

Table 1 Simulations of Confocal Images with Varying Densities of Green NPs

Tables Icon

Table 2 Simulations of 50 nm Diameter NPs, without Crosstalk and Noise

Tables Icon

Table 3 Simulations of 50 nm Diameter NPs, with Crosstalk and Noise Added

Tables Icon

Table 4 Simulations of 50 nm NPs without Crosstalk and Noise, with Varying Resolution

Equations (28)

Equations on this page are rendered with MathJax. Learn more.

I red+CT = I red +f I green
I reduced = I orig F× I CT .
G CC (x,y)= δ i r (x'+x,y'+y)δ i g (x',y') i r (x',y') i g (x',y')
G AC,r (x,y)= δ i r (x'+x,y'+y)δ i r (x',y') i r (x',y') i r (x',y') .
G fit (x,y)=G(0) e x 2 + y 2 σ + G
G(k,l)= 1 (Nk)(Nl) m=1 Nk n=1 Nl δ i s (m+k,n+l)δ i t (n,m) 1 N 2 n=1,m=1 N i s (m,n) 1 N 2 n=1,m=1 N i t (m,n)
G(k,l)= N 2 F 1 [ F[ i s (m,n)] F [ i t (m,n)] ] n=1,m=1 N i s (m,n) n=1,m=1 N i t (m,n)
i g = I g = I g,tot ( 1 A p A g N pg )+ I g,CT
i r = I r = Q p N pr + I r,CT
δ i r δ i g =Δ( I g,tot ( 1 A p A g N pg ) )Δ( Q p N pr )= I g,tot A p A g Q p N pg N pr .
G CC (0)= I g,tot A p A g Q p N pg N pr Q p N pr ( I g,tot ( 1 A p A g N pg ) ) = A p A g A r ( 1 A p A g N pg )
G CC (0) A p A g A r
N pr = 1 G AC,r (0)
N pr = A r N p A = A r n
A r = 1 n G ac,r (0) .
A g = ( ω g ω r ) 2 A r = ( ω g ω r ) 2 1 n G ac,r (0) .
A p = 1 n ω g ω r [ G ac,r (0) G CC (0) + ω g ω r ] 1
d= 2 π 1 n 13 14 [ G ac,r (0) G CC (0) + 13 14 ] 1 2
d= 2 π 1 n [ G ac,r (0) G CC (0) +1 ] 1 2 .
d= 257 ± 12 nm.
d= 2 π ( 13/14 )π 270 2 0.22 = 244 nm      ( raw )
d= 2 π ( 13/14 )π 270 2 0.27 = 270 nm      ( crosstalk reduced )
d= 65 ± 26 nm
d= 76 ± 17 nm.
d= 51 ± 19 nm ( Confocal, crosstalk compensated and deconvoluted )
d= 59 ± 17 nm ( STED, crosstalk compensated and deconvoluted ).
d 2 π π 290 2 0.013  = 66 nm ( approximation, confocal )
d 2 π π 150 2 0.12  = 104 nm ( approximation, STED ).

Metrics