Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses

Johanna Bückers; Dominik Wildanger; Giuseppe Vicidomini; Lars Kastrup; Stefan W. Hell

doi:10.1364/OE.19.003130

Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses

Johanna Bückers, Dominik Wildanger, Giuseppe Vicidomini, Lars Kastrup, and Stefan W. Hell

Optics Express
Vol. 19,
Issue 4,
pp. 3130-3143
(2011)
•https://doi.org/10.1364/OE.19.003130

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Johanna Bückers, Dominik Wildanger, Giuseppe Vicidomini, Lars Kastrup, and Stefan W. Hell, "Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses," Opt. Express 19, 3130-3143 (2011)

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Related Topics
Table of Contents Category
- Microscopy
Optics & Photonics Topics
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The topics in this list come from the OSA Optics and Photonics Topics applied to this article.
Previously assigned OCIS codes
- Fluorescence microscopy (180.2520)
- Resolution (350.5730)

About this Article
History
- Original Manuscript: January 4, 2011
- Manuscript Accepted: January 18, 2011
- Published: February 2, 2011
Virtual Issues
Virtual Journal for Biomedical Optics Vol. 6, Iss. 3

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Open Access

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.

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References

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Publication

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

Aslund, N.

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.

Betzig, E.

Bock, H.

Bonifacino, J. S.

Boruah, B. R.

Bossi, M.

Boyarskiy, V. P.

Brand, L.

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

Bückers, J.

Carlsson, K.

Chinenov, Y.

Cotlet, M.

Davidson, M. W.

De Schryver, F. C.

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.

Dunsby, C.

Eggeling, C.

Egner, A.

Foerster, T.

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

Folling, J.

French, P. M. W.

Galbraith, C. G.

Galbraith, J. A.

Geisler, C.

Gensch, T.

Gillette, J.

Girirajan, T. P. K.

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. 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]

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]

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

Hess, H. F.

Hess, S. T.

Hofkens, J.

Hu, C. D.

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.

Jakobs, S.

Kamin, D.

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. 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]

Kennedy, G.

Kerppola, T. K.

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.

Liljeborg, A.

Lindwasser, O. W.

Lippincott-Schwartz, J.

Mason, M. D.

Maus, M.

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]

Mossberg, K.

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.

Okada, T.

Olenych, S.

Patterson, G. H.

Philip, J.

Rittweger, E.

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]

Rizzoli, S. O.

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.

Schonle, A.

Schönle, A.

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.

Shroff, H.

Sougrat, R.

Stiel, A. C.

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.

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.

Westphal, V.

White, H.

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.

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.

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.

Zinchuk, V.

Acta Histochem. Cytochem. (1)

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)

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)

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]

J. Microsc. (4)

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]

Mol. Cell (1)

Nano Lett. (1)

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)

Nature (1)

Opt. Express (2)

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[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).
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Fig. 1 Setup of the multi-lifetime/multi-color STED microscope.

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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).

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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).

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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.

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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.

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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.

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Equations (6)

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g_{j} = N_{g} [\sum_{k = 1}^{n} (A_{k} \frac{{IRF}_{j} \otimes e^{j t / τ_{k}} + c}{\sum_{l = 1}^{N} ({IRF}_{l} \otimes e^{l t / τ_{k}} + c)}) + (1 - \sum_{k = 1}^{n} A_{k}) \frac{b_{j}}{\sum_{l = 1}^{N} b_{l}}] .

y = E x .

y_{i j} (r) = \sum_{k = 1}^{N} ε_{i k} [g_{k j} (r) \otimes x_{k} (r)]

Y (r) = E X (r) .

X (r) = E^{- 1} Y (r)

E = (\begin{matrix} 1 & 0.12 \\ 0.26 & 1 \end{matrix}), E^{- 1} = (\begin{matrix} 1
                                       .03 & -0
                                       .124 \\ -0
                                       .268 & 1
                                       .03 \end{matrix})