Abstract

We describe a STED microscope optimized for colocalization experiments with up to three colors. Two fluorescence labels are separated by their fluorescence lifetime whereas a third channel is discriminated by the wavelength of fluorescence emission. Since it does not require a second STED beam, separating by lifetime is insensitive to drift and thus optimally suited for colocalization analyses. Furthermore, we propose a setup having a second STED beam for long duration multicolor recording.

© 2011 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. T. Foerster, “Intermolecular energy migration and fluorescence,” Ann. Phys. 2, 55 (1948).
  2. P. Wu and L. Brand, “Resonance energy transfer: methods and applications,” Anal. Biochem. 218(1), 1–13 (1994).
    [CrossRef] [PubMed]
  3. C. D. Hu, Y. Chinenov, and T. K. Kerppola, “Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation,” Mol. Cell 9(4), 789–798 (2002).
    [CrossRef] [PubMed]
  4. T. K. Kerppola, “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells,” Nat. Protoc. 1(3), 1278–1286 (2006).
    [CrossRef]
  5. K. Carlsson and A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. 185(1), 37–46 (1997).
    [CrossRef]
  6. R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
    [CrossRef] [PubMed]
  7. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
    [CrossRef] [PubMed]
  8. V. Zinchuk, O. Zinchuk, and T. Okada, “Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena,” Acta Histochem. Cytochem. 40(4), 101–111 (2007).
    [CrossRef] [PubMed]
  9. S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
    [CrossRef] [PubMed]
  10. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [CrossRef] [PubMed]
  11. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
    [CrossRef] [PubMed]
  12. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
    [CrossRef] [PubMed]
  13. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
    [CrossRef] [PubMed]
  14. M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
    [CrossRef] [PubMed]
  15. A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
    [CrossRef] [PubMed]
  16. S. van de Linde, R. Kasper, M. Heilemann, and M. Sauer, “Photoswitching microscopy with standard fluorophores,” Appl. Phys. B 93(4), 725–731 (2008).
    [CrossRef]
  17. W. Heisenberg, The Physical Principles of the Quantum Theory (Chicago University Press, 1930).
  18. M. Bates, B. Huang, G. T. Dempsey, and X. W. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
    [CrossRef] [PubMed]
  19. G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
    [CrossRef] [PubMed]
  20. H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
    [CrossRef] [PubMed]
  21. M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
    [CrossRef] [PubMed]
  22. V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
    [CrossRef] [PubMed]
  23. J. Engelhardt, J. Keller, P. Hoyer, M. Reuss, T. Staudt, and S. W. Hell, “Molecular Orientation Affects Localization Accuracy in Superresolution Far-Field Fluorescence Microscopy,” Nano Lett. (2010).
  24. D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
    [CrossRef] [PubMed]
  25. D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
    [CrossRef] [PubMed]
  26. E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
    [CrossRef] [PubMed]
  27. M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
    [CrossRef] [PubMed]
  28. K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
    [CrossRef] [PubMed]
  29. R. Neher and E. Neher, “Optimizing imaging parameters for the separation of multiple labels in a fluorescence image,” J. Microsc. 213(1), 46–62 (2004).
    [CrossRef]
  30. D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
    [CrossRef]

2010 (1)

D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
[CrossRef]

2009 (2)

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
[CrossRef] [PubMed]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

2008 (5)

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and 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, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

S. van de Linde, R. Kasper, M. Heilemann, and M. Sauer, “Photoswitching microscopy with standard fluorophores,” Appl. Phys. B 93(4), 725–731 (2008).
[CrossRef]

2007 (6)

M. Bates, B. Huang, G. T. Dempsey, and X. W. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

V. Zinchuk, O. Zinchuk, and T. Okada, “Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena,” Acta Histochem. Cytochem. 40(4), 101–111 (2007).
[CrossRef] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

2006 (5)

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

T. K. Kerppola, “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells,” Nat. Protoc. 1(3), 1278–1286 (2006).
[CrossRef]

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

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

2004 (1)

R. Neher and E. Neher, “Optimizing imaging parameters for the separation of multiple labels in a fluorescence image,” J. Microsc. 213(1), 46–62 (2004).
[CrossRef]

2002 (2)

C. D. Hu, Y. Chinenov, and T. K. Kerppola, “Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation,” Mol. Cell 9(4), 789–798 (2002).
[CrossRef] [PubMed]

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

2001 (1)

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

1997 (1)

K. Carlsson and A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. 185(1), 37–46 (1997).
[CrossRef]

1994 (3)

P. Wu and L. Brand, “Resonance energy transfer: methods and applications,” Anal. Biochem. 218(1), 1–13 (1994).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[CrossRef] [PubMed]

1948 (1)

T. Foerster, “Intermolecular energy migration and fluorescence,” Ann. Phys. 2, 55 (1948).

Andresen, M.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Aslund, N.

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Auksorius, E.

Bates, M.

M. Bates, B. Huang, G. T. Dempsey, and X. W. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Belov, V. N.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

Betzig, E.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

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

Bock, H.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[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, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Boruah, B. R.

Bossi, M.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

Boyarskiy, V. P.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

Brand, L.

P. Wu and L. Brand, “Resonance energy transfer: methods and applications,” Anal. Biochem. 218(1), 1–13 (1994).
[CrossRef] [PubMed]

Bückers, J.

D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
[CrossRef]

Carlsson, K.

K. Carlsson and A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. 185(1), 37–46 (1997).
[CrossRef]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Chinenov, Y.

C. D. Hu, Y. Chinenov, and T. K. Kerppola, “Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation,” Mol. Cell 9(4), 789–798 (2002).
[CrossRef] [PubMed]

Cotlet, M.

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Davidson, M. W.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

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

De Schryver, F. C.

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Dempsey, G. T.

M. Bates, B. Huang, G. T. Dempsey, and X. W. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

Donnert, G.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

Dunsby, C.

Eggeling, C.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Egner, A.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Foerster, T.

T. Foerster, “Intermolecular energy migration and fluorescence,” Ann. Phys. 2, 55 (1948).

Folling, J.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

French, P. M. W.

Galbraith, C. G.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

Galbraith, J. A.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

Geisler, C.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Gensch, T.

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Gillette, J.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

Girirajan, T. P. K.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Heilemann, M.

S. van de Linde, R. Kasper, M. Heilemann, and M. Sauer, “Photoswitching microscopy with standard fluorophores,” Appl. Phys. B 93(4), 725–731 (2008).
[CrossRef]

Hell, S. W.

D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
[CrossRef]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and 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, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

S. W. Hell and 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, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Hofkens, J.

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Hu, C. D.

C. D. Hu, Y. Chinenov, and T. K. Kerppola, “Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation,” Mol. Cell 9(4), 789–798 (2002).
[CrossRef] [PubMed]

Huang, B.

M. Bates, B. Huang, G. T. Dempsey, and X. W. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

Jahn, R.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Jakobs, S.

D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
[CrossRef]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Kamin, D.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Kasper, R.

S. van de Linde, R. Kasper, M. Heilemann, and M. Sauer, “Photoswitching microscopy with standard fluorophores,” Appl. Phys. B 93(4), 725–731 (2008).
[CrossRef]

Kastrup, L.

D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
[CrossRef]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

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

Keller, J.

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

Kennedy, G.

Kerppola, T. K.

T. K. Kerppola, “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells,” Nat. Protoc. 1(3), 1278–1286 (2006).
[CrossRef]

C. D. Hu, Y. Chinenov, and T. K. Kerppola, “Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation,” Mol. Cell 9(4), 789–798 (2002).
[CrossRef] [PubMed]

Lanigan, P. M. P.

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Lauterbach, M. A.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Liljeborg, A.

K. Carlsson and A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. 185(1), 37–46 (1997).
[CrossRef]

Lindwasser, O. W.

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

Mason, M. D.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Maus, M.

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Medda, R.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Mossberg, K.

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Neher, E.

R. Neher and E. Neher, “Optimizing imaging parameters for the separation of multiple labels in a fluorescence image,” J. Microsc. 213(1), 46–62 (2004).
[CrossRef]

Neher, R.

R. Neher and E. Neher, “Optimizing imaging parameters for the separation of multiple labels in a fluorescence image,” J. Microsc. 213(1), 46–62 (2004).
[CrossRef]

Neil, M. A. A.

Neumann, D.

D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
[CrossRef]

Okada, T.

V. Zinchuk, O. Zinchuk, and T. Okada, “Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena,” Acta Histochem. Cytochem. 40(4), 101–111 (2007).
[CrossRef] [PubMed]

Olenych, S.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

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

Philip, J.

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Rittweger, E.

Rizzoli, S. O.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Sauer, M.

S. van de Linde, R. Kasper, M. Heilemann, and M. Sauer, “Photoswitching microscopy with standard fluorophores,” Appl. Phys. B 93(4), 725–731 (2008).
[CrossRef]

Schaffer, J.

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Schonle, A.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

Schönle, A.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

Seidel, C. A.

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

Shroff, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[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, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Stiel, A. C.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

van de Linde, S.

S. van de Linde, R. Kasper, M. Heilemann, and M. Sauer, “Photoswitching microscopy with standard fluorophores,” Appl. Phys. B 93(4), 725–731 (2008).
[CrossRef]

von Middendorff, C.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Wenzel, D.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

Westphal, V.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

White, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

Wichmann, J.

Wildanger, D.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

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

Willig, K. I.

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Wu, P.

P. Wu and L. Brand, “Resonance energy transfer: methods and applications,” Anal. Biochem. 218(1), 1–13 (1994).
[CrossRef] [PubMed]

Wurm, C. A.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

Zhuang, X. W.

M. Bates, B. Huang, G. T. Dempsey, and X. W. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Zinchuk, O.

V. Zinchuk, O. Zinchuk, and T. Okada, “Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena,” Acta Histochem. Cytochem. 40(4), 101–111 (2007).
[CrossRef] [PubMed]

Zinchuk, V.

V. Zinchuk, O. Zinchuk, and T. Okada, “Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena,” Acta Histochem. Cytochem. 40(4), 101–111 (2007).
[CrossRef] [PubMed]

Acta Histochem. Cytochem. (1)

V. Zinchuk, O. Zinchuk, and T. Okada, “Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena,” Acta Histochem. Cytochem. 40(4), 101–111 (2007).
[CrossRef] [PubMed]

Anal. Biochem. (1)

P. Wu and L. Brand, “Resonance energy transfer: methods and applications,” Anal. Biochem. 218(1), 1–13 (1994).
[CrossRef] [PubMed]

Anal. Chem. (1)

M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules,” Anal. Chem. 73(9), 2078–2086 (2001).
[CrossRef] [PubMed]

Ann. Phys. (1)

T. Foerster, “Intermolecular energy migration and fluorescence,” Ann. Phys. 2, 55 (1948).

Appl. Phys. B (1)

S. van de Linde, R. Kasper, M. Heilemann, and M. Sauer, “Photoswitching microscopy with standard fluorophores,” Appl. Phys. B 93(4), 725–731 (2008).
[CrossRef]

Biophys. J. (4)

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, “Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters,” Biophys. J. 93(9), 3285–3290 (2007).
[CrossRef] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

J. Microsc. (4)

K. Carlsson, N. Aslund, K. Mossberg, J. Philip, M. Maus, M. Cotlet, J. Hofkens, T. Gensch, F. C. De Schryver, J. Schaffer, and C. A. Seidel, “Simultaneous confocal recording of multiple fluorescent labels with improved channel separation,” J. Microsc. 176(Pt 3), 287–299 (1994).
[CrossRef] [PubMed]

R. Neher and E. Neher, “Optimizing imaging parameters for the separation of multiple labels in a fluorescence image,” J. Microsc. 213(1), 46–62 (2004).
[CrossRef]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

K. Carlsson and A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. 185(1), 37–46 (1997).
[CrossRef]

Mol. Cell (1)

C. D. Hu, Y. Chinenov, and T. K. Kerppola, “Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation,” Mol. Cell 9(4), 789–798 (2002).
[CrossRef] [PubMed]

Nano Lett. (1)

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, “Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species,” Nano Lett. 8(8), 2463–2468 (2008).
[CrossRef] [PubMed]

Nat. Methods (2)

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Nat. Protoc. (1)

T. K. Kerppola, “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells,” Nat. Protoc. 1(3), 1278–1286 (2006).
[CrossRef]

Nature (1)

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (2)

PMC Biophys (1)

D. Neumann, J. Bückers, L. Kastrup, S. W. Hell, and S. Jakobs, “Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms,” PMC Biophys 3(1), 1–4 (2010).
[CrossRef]

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

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[CrossRef] [PubMed]

Science (3)

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

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

M. Bates, B. Huang, G. T. Dempsey, and X. W. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

Other (2)

W. Heisenberg, The Physical Principles of the Quantum Theory (Chicago University Press, 1930).

J. Engelhardt, J. Keller, P. Hoyer, M. Reuss, T. Staudt, and S. W. Hell, “Molecular Orientation Affects Localization Accuracy in Superresolution Far-Field Fluorescence Microscopy,” Nano Lett. (2010).

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 (6)

Fig. 1
Fig. 1

Setup of the multi-lifetime/multi-color STED microscope.

Fig. 2
Fig. 2

Fluorescence separation by lifetime analysis. Images of tubulin labeled with ATTO 647N (left) or KK 114 (right) were recorded by TCPSC (A). A per-pixel monoexponential fit of the lifetime histogram reproduces the original images with residuals < 10% (B, C). Even after adding the photocounts of the original recordings (D) the two channels can be recovered by fitting a biexponential function to the lifetime histograms: (E) shows the amplitude images (top) of the two lifetime components along with the corresponding difference to the initial data (bottom).

Fig. 3
Fig. 3

Two-channel STED imaging by fluorescence lifetime separation. Tubulin and lamin were immunostained with ATTO 647N and KK 114, respectively. (A) Raw intensity STED data and (B) channels decomposed by lifetime separation (green: tubulin, red: lamin).

Fig. 4
Fig. 4

Combined spectral and lifetime separation. Lamin and tubulin were stained with KK 114 and ATTO 647N, respectively, which were segregated by lifetime analysis. The ATTO 590 fluorescence (clathrin) was spectrally separated from those of the other dyes.

Fig. 5
Fig. 5

Improved beam path configuration for dual-wavelength STED imaging. Contrary to the previous design the STED beams are jointly coupled to a single mode optical fiber and are guided to the same phase plate.

Fig. 6
Fig. 6

. Stability of the dual-wavelength colocalization measurements. The main scatter plot shows the offset of individual beads between the two color channels at various times. The inset shows the image offset as obtained by averaging over the individual offsets. Over the whole measurement period the offset remains constant to within the statistical error of the experiment.

Equations (6)

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

g j = N g [ k = 1 n ( A k IRF j e j t / τ k + c l = 1 N ( IRF l e l t / τ k + c ) ) + ( 1 k = 1 n A k ) b j l = 1 N b l ] .
y = E x   .
y i j ( r ) = k = 1 N ε i k [ g k j ( r ) x k ( r ) ]
Y ( r ) = E X ( r ) .
X ( r ) = E 1 Y ( r )
E = ( 1 0.12 0.26 1 ) , E 1 = ( 1 .03 -0 .124 -0 .268 1 .03 )

Metrics