Abstract

Diffraction-unlimited resolution provided by Stimulated Emission Depletion (STED) microscopy allows for imaging cellular processes in living cells that are not visible by conventional microscopy. However, it has so far not been possible to study dynamic nanoscale interactions because multicolor live cell STED microscopy has yet to be demonstrated and suitable labeling technologies and protocols are lacking. Here we report the first realization of two-color STED imaging in living cells. Using improved SNAPf and CLIPf technologies to label epidermal growth factor (EGF) and EGF receptor (EGFR), we report resolutions of 78 nm and 82 nm for 22 sequential two-color scans in living cells.

© 2011 OSA

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]

2011 (2)

2010 (6)

K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
[CrossRef] [PubMed]

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[CrossRef]

2009 (4)

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

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

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

2008 (4)

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (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]

2007 (1)

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]

2006 (3)

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]

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

2004 (1)

A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson, “Labeling of fusion proteins with synthetic fluorophores in live cells,” Proc. Natl. Acad. Sci. U.S.A. 101(27), 9955–9959 (2004).
[CrossRef] [PubMed]

2003 (1)

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[CrossRef] [PubMed]

1998 (1)

R. E. Carter and A. Sorkin, “Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera,” J. Biol. Chem. 273(52), 35000–35007 (1998).
[CrossRef] [PubMed]

1994 (1)

1991 (1)

A. Sorkin and G. Carpenter, “Dimerization of internalized epidermal growth factor receptors,” J. Biol. Chem. 266(34), 23453–23460 (1991).
[PubMed]

Arrivoli, C.

A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson, “Labeling of fusion proteins with synthetic fluorophores in live cells,” Proc. Natl. Acad. Sci. U.S.A. 101(27), 9955–9959 (2004).
[CrossRef] [PubMed]

Bachmann, J.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Baker, B.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Bates, M.

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

Beaufils, F.

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[CrossRef] [PubMed]

Belov, V. N.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[CrossRef]

Berget, P. B.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[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, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bewersdorf, J.

T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express 19(14), 13351–13357 (2011).
[CrossRef] [PubMed]

K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[CrossRef] [PubMed]

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
[CrossRef] [PubMed]

Bhattacharyya, D.

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
[CrossRef] [PubMed]

Bierwagen, J.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[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]

Bruchez, M. P.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Bückers, J.

Buswell, J.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Carpenter, G.

A. Sorkin and G. Carpenter, “Dimerization of internalized epidermal growth factor receptors,” J. Biol. Chem. 266(34), 23453–23460 (1991).
[PubMed]

Carter, R. E.

R. E. Carter and A. Sorkin, “Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera,” J. Biol. Chem. 273(52), 35000–35007 (1998).
[CrossRef] [PubMed]

Corrêa, I. R.

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[CrossRef] [PubMed]

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[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, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Donnert, G.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (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]

Dyba, M.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Eggeling, C.

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[CrossRef]

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[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]

Egner, A.

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Ehrhard, T.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Engelhardt, J.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Fitzpatrick, J. A. J.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Gautier, A.

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[CrossRef] [PubMed]

Gendreizig, S.

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[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]

Glick, B. S.

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
[CrossRef] [PubMed]

Gould, T. J.

T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express 19(14), 13351–13357 (2011).
[CrossRef] [PubMed]

K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[CrossRef] [PubMed]

Gronemeyer, T.

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[CrossRef] [PubMed]

Harke, B.

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[CrossRef]

Hein, B.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
[CrossRef] [PubMed]

Heinis, C.

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[CrossRef] [PubMed]

Hell, S. W.

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[CrossRef] [PubMed]

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[CrossRef]

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

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

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (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]

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]

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]

Herten, D. P.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[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]

Howard, A.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[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]

Jakobs, S.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (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]

Johnsson, K.

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[CrossRef] [PubMed]

A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson, “Labeling of fusion proteins with synthetic fluorophores in live cells,” Proc. Natl. Acad. Sci. U.S.A. 101(27), 9955–9959 (2004).
[CrossRef] [PubMed]

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[CrossRef] [PubMed]

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Juillerat, A.

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[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]

Kastrup, L.

Keenan, R. J.

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
[CrossRef] [PubMed]

Keller, J.

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]

Keppler, A.

A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson, “Labeling of fusion proteins with synthetic fluorophores in live cells,” Proc. Natl. Acad. Sci. U.S.A. 101(27), 9955–9959 (2004).
[CrossRef] [PubMed]

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[CrossRef] [PubMed]

Kindermann, M.

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
[CrossRef] [PubMed]

Klingmüller, U.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Kolmakov, K.

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[CrossRef]

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[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]

Leutenegger, M.

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[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]

Lymperopoulos, K.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Masharina, A.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[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]

Maurel, D.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Medda, R.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

Meyer, L.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

Morozova, K. S.

K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[CrossRef] [PubMed]

Müller, V.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

Myers, J. R.

Noren, C. J.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Olenych, 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]

Öz, S.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[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]

Pfeifer, A. C.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Piatkevich, K. D.

K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[CrossRef] [PubMed]

Pick, H.

A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson, “Labeling of fusion proteins with synthetic fluorophores in live cells,” Proc. Natl. Acad. Sci. U.S.A. 101(27), 9955–9959 (2004).
[CrossRef] [PubMed]

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[CrossRef] [PubMed]

Punge, A.

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

Ringemann, C.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

Rizzoli, S. O.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (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]

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]

Rust, M. J.

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

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (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]

Schulmeister, S.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Schwarz, U.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Sieber, J. J.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Siegberg, D.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
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R. E. Carter and A. Sorkin, “Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera,” J. Biol. Chem. 273(52), 35000–35007 (1998).
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A. Sorkin and G. Carpenter, “Dimerization of internalized epidermal growth factor receptors,” J. Biol. Chem. 266(34), 23453–23460 (1991).
[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]

Sourjik, V.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Stöhr, K.

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Strack, R. L.

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
[CrossRef] [PubMed]

Sun, L.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Sun, X.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Szent-Gyorgyi, C.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
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D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
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K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[CrossRef] [PubMed]

Vicidomini, G.

Vogel, H.

A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson, “Labeling of fusion proteins with synthetic fluorophores in live cells,” Proc. Natl. Acad. Sci. U.S.A. 101(27), 9955–9959 (2004).
[CrossRef] [PubMed]

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[CrossRef] [PubMed]

Waggoner, A. S.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Westphal, V.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and 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, 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]

Wichmann, J.

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J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

Willig, K. I.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Woolford, C. A.

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Wurm, C. A.

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
[CrossRef]

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (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]

Xu, M.-Q.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

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J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Zhang, A.

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Zhang, J.

K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[CrossRef] [PubMed]

Zhuang, X.

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

Anal. Chem. (1)

K. Stöhr, D. Siegberg, T. Ehrhard, K. Lymperopoulos, S. Öz, S. Schulmeister, A. C. Pfeifer, J. Bachmann, U. Klingmüller, V. Sourjik, and D. P. Herten, “Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background,” Anal. Chem. 82(19), 8186–8193 (2010).
[CrossRef] [PubMed]

Annu. Rev. Cell Dev. Biol. (1)

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
[CrossRef] [PubMed]

Biochemistry (1)

R. L. Strack, B. Hein, D. Bhattacharyya, S. W. Hell, R. J. Keenan, and B. S. Glick, “A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling,” Biochemistry 48(35), 8279–8281 (2009).
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Bioconjug. Chem. (1)

J. A. J. Fitzpatrick, Q. Yan, J. J. Sieber, M. Dyba, U. Schwarz, C. Szent-Gyorgyi, C. A. Woolford, P. B. Berget, A. S. Waggoner, and M. P. Bruchez, “STED nanoscopy in living cells using Fluorogen Activating Proteins,” Bioconjug. Chem. 20(10), 1843–1847 (2009).
[CrossRef] [PubMed]

Biophys. J. (4)

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
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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).
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K. S. Morozova, K. D. Piatkevich, T. J. Gould, J. Zhang, J. Bewersdorf, and V. V. Verkhusha, “Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy,” Biophys. J. 99(2), L13–L15 (2010).
[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).
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Chem. Biol. (1)

A. Gautier, A. Juillerat, C. Heinis, I. R. Corrêa, M. Kindermann, F. Beaufils, and K. Johnsson, “An engineered protein tag for multiprotein labeling in living cells,” Chem. Biol. 15(2), 128–136 (2008).
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ChemBioChem (1)

X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard, J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren, M.-Q. Xu, and I. R. Corrêa., “Development of SNAP-tag fluorogenic probes for wash-free fluoescence imaging,” ChemBioChem . in press.
[PubMed]

Chemistry (1)

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

Eur. J. Org. Chem. (1)

K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, and S. W. Hell, “A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy,” Eur. J. Org. Chem. 2010(19), 3593–3610 (2010).
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Nano Lett. (1)

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson, “A general method for the covalent labeling of fusion proteins with small molecules in vivo,” Nat. Biotechnol. 21(1), 86–89 (2003).
[CrossRef] [PubMed]

Nat. Methods (3)

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

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

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

Opt. Express (2)

Opt. Lett. (1)

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

A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson, “Labeling of fusion proteins with synthetic fluorophores in live cells,” Proc. Natl. Acad. Sci. U.S.A. 101(27), 9955–9959 (2004).
[CrossRef] [PubMed]

Science (2)

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]

Small (1)

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

Schematic of experimental labeling procedure using SNAPf or CLIPf reactions to specifically label EGFR and EGF. (A) In living cells, SNAPf recognizes its substrate, BG, and undergoes a Sn2 type reaction that displaces BG, resulting in a permanent covalent bond between the EGFR and Chromeo494 (green star). (B) EGF-CLIPf is recombinantly expressed and purified, labeled with BC-ATTO647N (BC-647N) producing exogenous EGF-CLIPf-ATTO647N. (C) Live cells expressing EGFR-SNAPf are labeled with Chromeo494, followed by incubation with EGF-CLIPf-ATTO647N. This results in the double labeling of the EGFR-EGR complex for two-color STED imaging. EGFR is known to form a homodimer upon ligand binding, but is shown as a single receptor in this schematic for clarity. Cyto = cytoplasm, PM = plasma membrane and ECM = extracellular matrix.

Fig. 2
Fig. 2

Resolution measurements for ATTO647N and Chromeo494 in living HEK293 cells. HEK293 cells were treated with 100 ng/mL EGF-CLIPf-ATTO647N and imaged with (A) STED and (B) confocal microscopy. The area depicted by the white box (A) is shown in (C) STED and (D) confocal. The boxes in (C) and (D) were used to generate the line profiles for ATTO647N in (E) and (F). HEK293 cells stably expressing EGFR-SNAPf were treated with 10 μM BG-Chromeo494 for 10 min at 37°C and imaged by (G) STED and (H) confocal microscopy. The area depicted by the white box (G) for Chromeo494 is shown in (I) STED and (J) confocal. The boxes in (I) and (J) were used to generate the line profiles for Chromeo494 in (K) STED and (L) confocal. Solid red lines indicate the Lorentzian (STED) and Gaussian (confocal) least squares fits used to determine the full width at half-maximum (FWHM) values.

Fig. 3
Fig. 3

Live cell two-color STED time series of HEK293 cells labeled with EGF-CLIPf-ATTO647N (magenta) and EGFR-SNAPf-Chromeo494 (green). Data has been normalized to correct for bleaching. The shown images have been cropped from the original raw data. Scale bar = 1 μm.

Tables (1)

Tables Icon

Table 1 Fluorophores Evaluated for Two-Color Live Cell STED Microscopy Experiments by Excitation Maximum (λex), Depletion Wavelength (λSTED) and Binding Characteristics

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