Abstract

We report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable (fluorescence) state transitions induced by a spatially modulated light pattern. The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce. By switching fluorophores off only if there is an adjacent fluorescent feature to be recorded, the method reduces the number of state transitions as well as the average time a dye is forced to reside in an off-state. Thus, the photobleaching of the sample is reduced, while resolution and recording speed are preserved. The power of the method is exemplified by imaging immunolabeled glial cells with up to 8-fold reduced photobleaching.

© 2011 OSA

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    [CrossRef]
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    [CrossRef]

2009 (3)

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

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

R. Heintzmann and M. G. L. Gustafsson, “Subdiffraction resolution in continuous samples,” Nat. Photonics 3(7), 362–364 (2009).
[CrossRef]

2008 (4)

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schoenle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots,” Angew. Chem. Int. Ed. Engl. 47(14), 2685–2688 (2008).
[CrossRef] [PubMed]

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

2007 (5)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

2006 (4)

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. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (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]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

2005 (2)

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

2004 (1)

S. W. Hell, “Strategy for far-field optical imaging and writing without diffraction limit,” Phys. Lett. A 326(1-2), 140–145 (2004).
[CrossRef]

2003 (1)

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

2002 (1)

2001 (1)

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

2000 (2)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

1996 (1)

L. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

1995 (1)

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

1994 (1)

1990 (1)

C. G. Dotti and K. Simons, “Polarized Sorting of viral glycoproteins to the axon and dendrites of hippocampal-neurons in culture,” Cell 62, 63–72 (1990).
[CrossRef] [PubMed]

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Back, S.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Bates, M.

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]

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]

Beyreuther, K.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Bock, H.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

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]

Bossi, M.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Bretschneider, S.

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

Cena, V.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Comella, J. X.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Cremer, C.

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]

Dhonukshe, P. B.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

Dittrich, P. S.

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

Donnert, G.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Dotti, C. G.

C. G. Dotti and K. Simons, “Polarized Sorting of viral glycoproteins to the axon and dendrites of hippocampal-neurons in culture,” Cell 62, 63–72 (1990).
[CrossRef] [PubMed]

Dyba, M.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Eggeling, C.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Egner, A.

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Encinas, M.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Engelhardt, J.

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots,” Angew. Chem. Int. Ed. Engl. 47(14), 2685–2688 (2008).
[CrossRef] [PubMed]

Fölling, J.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Gadella, T. W. J.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

Gallego, C.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Geisler, C.

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

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]

Gruebl, T.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Gustafsson, M. G. L.

R. Heintzmann and M. G. L. Gustafsson, “Subdiffraction resolution in continuous samples,” Nat. Photonics 3(7), 362–364 (2009).
[CrossRef]

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Haas, P.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Han, K. Y.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Harke, B.

Heilemann, M.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Hein, B.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Heintzmann, R.

Hell, S. W.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

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

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots,” Angew. Chem. Int. Ed. Engl. 47(14), 2685–2688 (2008).
[CrossRef] [PubMed]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schoenle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

S. W. Hell, “Strategy for far-field optical imaging and writing without diffraction limit,” Phys. Lett. A 326(1-2), 140–145 (2004).
[CrossRef]

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

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]

Hoebe, R. A.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

Iglesias, M.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Irvine, S. E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots,” Angew. Chem. Int. Ed. Engl. 47(14), 2685–2688 (2008).
[CrossRef] [PubMed]

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Jakobs, S.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Jovin, T. M.

Kasper, R.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Keller, J.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schoenle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Kins, S.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Kirsch, J.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Kroug, M.

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[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]

Liu, Y. H.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Luhrmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Manders, E. M. M.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

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]

Medda, R.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Mueller, U.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Muhaisen, A.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, 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]

Person, B.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Rittweger, E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots,” Angew. Chem. Int. Ed. Engl. 47(14), 2685–2688 (2008).
[CrossRef] [PubMed]

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (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.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Schoenle, A.

Schonle, A.

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

Schwille, P.

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

Simons, K.

C. G. Dotti and K. Simons, “Polarized Sorting of viral glycoproteins to the axon and dendrites of hippocampal-neurons in culture,” Cell 62, 63–72 (1990).
[CrossRef] [PubMed]

Song, L. L.

L. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[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]

Staudt, T.

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots,” Angew. Chem. Int. Ed. Engl. 47(14), 2685–2688 (2008).
[CrossRef] [PubMed]

Steinhauer, C.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Tanke, H. J.

L. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

Tinnefeld, P.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Tschaepe, J. A.

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

Ullal, C. K.

Van Noorden, C. J. F.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

Van Oven, C. H.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

Varma, C. A.

L. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

Verhoeven, J. W.

L. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

Vogelsang, J.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Von Middendorff, C.

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

Wang, H. Y.

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

Westphal, V.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schoenle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

Wichmann, J.

Wurm, C. A.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

Zhuang, X. W.

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]

Angew. Chem. Int. Ed. Engl. (2)

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct light-driven modulation of luminescence from Mn-doped ZnSe quantum dots,” Angew. Chem. Int. Ed. Engl. 47(14), 2685–2688 (2008).
[CrossRef] [PubMed]

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem. Int. Ed. Engl. 47(29), 5465–5469 (2008).
[CrossRef] [PubMed]

Appl. Phys. B (3)

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

H. Bock, C. Geisler, C. A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[CrossRef]

Biophys. J. (2)

L. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[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]

Cell (1)

C. G. Dotti and K. Simons, “Polarized Sorting of viral glycoproteins to the axon and dendrites of hippocampal-neurons in culture,” Cell 62, 63–72 (1990).
[CrossRef] [PubMed]

J. Neurochem. (1)

M. Encinas, M. Iglesias, Y. H. Liu, H. Y. Wang, A. Muhaisen, V. Cena, C. Gallego, and J. X. Comella, “Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells,” J. Neurochem. 75(3), 991–1003 (2000).
[CrossRef] [PubMed]

J. Neurosci. Res. (1)

S. Back, P. Haas, J. A. Tschaepe, T. Gruebl, J. Kirsch, U. Mueller, K. Beyreuther, and S. Kins, “beta-amyloid precursor protein can be transported independent of any sorting signal to the axonal and dendritic compartment,” J. Neurosci. Res. 85(12), 2580–2590 (2007).
[CrossRef] [PubMed]

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

Nat. Biotechnol. (1)

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

Nat. Methods (3)

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]

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

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Nat. Photonics (2)

R. Heintzmann and M. G. L. Gustafsson, “Subdiffraction resolution in continuous samples,” Nat. Photonics 3(7), 362–364 (2009).
[CrossRef]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Nature Biotechnol. (1)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnol. 25, 249–253 (2007).
[CrossRef]

Opt. Express (1)

Opt. Lett. (1)

Phys. Lett. A (1)

S. W. Hell, “Strategy for far-field optical imaging and writing without diffraction limit,” Phys. Lett. A 326(1-2), 140–145 (2004).
[CrossRef]

Phys. Rev. Lett. (2)

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (3)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Science (2)

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. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

Other (1)

S. Hell, “Far-Field Optical Nanoscopy,” in Single Molecule Spectroscopy in Chemistry, A. Gräslund, Rigler, R., Widengren, J., ed. (Springer, Berlin, 2009), pp. 365 - 398.

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Figures (6)

Fig. 1
Fig. 1

Exposure decision-making process for RESCue-STED. a) The role of the doughnut-shaped STED beam is to switch off the fluorescence capability of molecules or features residing within the diffraction barrier, so that the ones located at about the doughnut zero are allowed to emit and hence be registered independently from their neighbors right outside the ‘fluorescence-allowed’ region. The effective PSF (blue line) describes the region in which the molecules can fluoresce. The red line illustrates the reduced number of transition cycles through RESCue with dT/pT = 0.1. The idea behind RESCue-STED is to avoid extended optical forcing of molecules to reside in the ground (off) state by stimulated emission if there is no feature or molecule to be recorded within the subdiffraction sized region given by the effective PSF. b). Two objects falling within subdiffraction distances are separated by switching the fluorescence of one of them off; here this is accomplished by the red doughnut-shaped STED beam (large red circle). If no fluorescent feature is present within the subdiffraction fluorescence area (small white circle), the optical beams can be interrupted, because there is no need for the STED beam to switch off features for separation. In this way the number of state transition cycles and hence the bleaching probability is reduced. Grey circles indicate fluorophores. The grid marks the pixels.

Fig. 2
Fig. 2

Setup for RESCue-STED (left side). AOTFs are added to the STED microscope which are used to switch both laser beams on and off during the RESCue experiments depending on the measured fluorescence photon flux at each position in the sample. Three cases have to be considered (right side): Case 1: The lower threshold is not reached during the decision time dT (no fluorescent object is present in the reduced STED focus). The lasers are switched off during the dwell time pT. Case 2: The lTh is reached during dT (a fluorescent object is present in the effective STED focus). The lasers remain switched on during pT. Case 3: The lower and the upper threshold uTh is reached during the dT and the readout time rT respectively (a bright object is detected). The lasers are switched off after a certain number of photons are collected according to uTh. DM: dichroic mirror.

Fig. 3
Fig. 3

In RESCue mode bleaching is reduced 5-fold compared to standard STED, exemplified by recording fluorescent beads. (a) and (c) are confocal references taken before and after a series of 10 STED images (b). Each series differs in RESCue settings: (standard) RESCue disabled, (lTh) RESCue with a lower threshold lTh = 5 photons and dT = 50 µs, (lTh&uTh) RESCue with additional upper threshold uTh = 28 photons. The pixel dwell time pT = 400 µs for all scans. (d) The series with parameter as in (b) with the STED beam switched off show a 20% larger bleach factor than in the RESCue scan with lTh&uTh.

Fig. 4
Fig. 4

RESCue reduces photobleaching of Atto565 marked APP proteins in fixed primary mouse neurons by a factor of 4. Confocal image (a, left side) of the APP distribution and STED counterpart (raw data) (a, middle). The line profile reveals 50 nm resolution (a, right side). A confocal image is recorded before (b) and after three STED scans. Without RESCue (c, standard) bleaching precludes data acquisition. RESCue conditions preserve fluorescence better (lTh, lTh&uTh). Parameters: lTh=6 photons, uTh=25 photons and dT = 40 µs. Due to the sparse APP distribution the bleaching reduction is dominated by the lTh modality. pT = 500 µs for the confocal and 300 µs for the STED scans.

Fig. 5
Fig. 5

The parameter uTh further decreases Atto565 bleaching in immunostained glial fibrillary proteins (GFAP) in glioblastoma cells (u373), attaining an overall bleaching reduction factor of 8. Analogous to Fig. 4, the confocal images are recorded before (a) and after (c) a series of three STED scans. Without RESCue, almost all fluorescence was bleached after the third scan (b, standard). With a lower threshold lTh of 6 photons and a decision time dT of 40 µs, the fluorescence of the densely labeled filaments can be preserved for significantly more scans (b, lTh). Moreover, with an additional upper threshold uTh of 28 photons, the bleaching reduction can be further improved by a factor of 2 (b, lTh & uTh). The pixel dwell time pT was set to 500 µs for the confocal and 300 µs for the STED scans.

Fig. 6
Fig. 6

RESCue allows the measurement of ATTO647N immunostained nuclear lamina in neuroblastoma cells in three dimensions. The left image (STED) shows the attempt to image the lamina without RESCue but fails due to on-off switching fatigue of the fluorophores. Without RESCue the SNR is not sufficient for 3D rendering. The signal is to low and clipped with the background by the 3D reconstruction. An effective use of the limited state transition cycles through RESCue-STED enables 3D subdiffraction imaging (right hand side). The lengths of x, z, y coordinates represent 1 µm, 1 µm and 0.5 µm respectively.

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