Abstract

We report the development of a stimulated emission depletion (STED) selective plane illumination (SPIM) microscope based on a single diode-pumped solid state (DPSS) laser that simultaneously delivers nanosecond-pulses at two wavelengths. The two wavelengths, 355 nm and 532 nm, are generated by harmonic conversion and they are used to induce respectively excitation and stimulated emission depletion. This source should allow a low-cost, compact, very efficient and simplified STED scheme since the two beams are intrinsically aligned and synchronized. Using a chromatic beam shaping device which leaves the excitation beam unaffected and produces a donut-shaped STED beam, we demonstrate a 300% reduction of the light sheet thickness, together with an enhancement of the sheet uniformity over larger field of view, at low STED power, in Coumarin dye solution.

© 2014 Optical Society of America

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References

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2014 (2)

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

P. Zhang, P. M. Goodwin, and J. H. Werner, “Fast, super resolution imaging via Bessel-beam stimulated emission depletion microscopy,” Opt. Express 22(10), 12398–12409 (2014).
[Crossref] [PubMed]

2013 (1)

2011 (3)

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

M. Friedrich, Q. Gan, V. Ermolayev, and G. S. Harms, “STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution,” Biophys. J. 100(8), L43–L45 (2011).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

2010 (3)

2008 (2)

2007 (5)

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

N. Pollak, C. Dölle, and M. Ziegler, “The power to reduce: pyridine nucleotides - small molecules with a multitude of functions,” Biochem. J. 402(2), 205–218 (2007).
[Crossref] [PubMed]

2006 (2)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

2005 (1)

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4pi fluorescence microscopy: theory and experiment,” New J. Phys. 7, 134 (2005).
[Crossref]

2004 (1)

2003 (1)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[Crossref]

2002 (1)

M. Dyba and S. W. Hell, “Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[Crossref] [PubMed]

2000 (1)

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]

1994 (2)

B. Boulanger, M. M. Fejer, R. Blachman, and P. F. Bordui, “Study of KTiOPO gray-tracking at 1064, 532, and 355 nm,” Appl. Phys. Lett. 65(19), 2401–2403 (1994).
[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]

1992 (1)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[Crossref] [PubMed]

1974 (1)

E. Schimitschek, J. Trias, P. Hammond, and R. Atkins, “Laser performance and stability of fluorinated coumarin dyes,” Opt. Commun. 11(4), 352–355 (1974).
[Crossref]

Alessandri, K.

Andersen, P. E.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

Atkins, R.

E. Schimitschek, J. Trias, P. Hammond, and R. Atkins, “Laser performance and stability of fluorinated coumarin dyes,” Opt. Commun. 11(4), 352–355 (1974).
[Crossref]

Betzig, E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

Blachman, R.

B. Boulanger, M. M. Fejer, R. Blachman, and P. F. Bordui, “Study of KTiOPO gray-tracking at 1064, 532, and 355 nm,” Appl. Phys. Lett. 65(19), 2401–2403 (1994).
[Crossref]

Blanca, C. M.

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[Crossref]

Bordui, P. F.

B. Boulanger, M. M. Fejer, R. Blachman, and P. F. Bordui, “Study of KTiOPO gray-tracking at 1064, 532, and 355 nm,” Appl. Phys. Lett. 65(19), 2401–2403 (1994).
[Crossref]

Boulanger, B.

B. Boulanger, M. M. Fejer, R. Blachman, and P. F. Bordui, “Study of KTiOPO gray-tracking at 1064, 532, and 355 nm,” Appl. Phys. Lett. 65(19), 2401–2403 (1994).
[Crossref]

Cižmár, T.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Coll-Lladó, C.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Dalgarno, H. I.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Davidson, M. W.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

Dholakia, K.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Dölle, C.

N. Pollak, C. Dölle, and M. Ziegler, “The power to reduce: pyridine nucleotides - small molecules with a multitude of functions,” Biochem. J. 402(2), 205–218 (2007).
[Crossref] [PubMed]

Donnert, G.

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

Dyba, M.

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4pi fluorescence microscopy: theory and experiment,” New J. Phys. 7, 134 (2005).
[Crossref]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[Crossref]

M. Dyba and S. W. Hell, “Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[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]

Eggeling, C.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[Crossref] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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.

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]

Eickhoff, J.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

Eliceiri, K. W.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

Engelhardt, J.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

M. Reuss, J. Engelhardt, and S. W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation,” Opt. Express 18(2), 1049–1058 (2010).
[Crossref] [PubMed]

Ermolayev, V.

M. Friedrich, Q. Gan, V. Ermolayev, and G. S. Harms, “STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution,” Biophys. J. 100(8), L43–L45 (2011).
[Crossref] [PubMed]

Fahrbach, F. O.

Fejer, M. M.

B. Boulanger, M. M. Fejer, R. Blachman, and P. F. Bordui, “Study of KTiOPO gray-tracking at 1064, 532, and 355 nm,” Appl. Phys. Lett. 65(19), 2401–2403 (1994).
[Crossref]

Ferrier, D. E.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Flyvbjerg, H.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Friedrich, M.

M. Friedrich, Q. Gan, V. Ermolayev, and G. S. Harms, “STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution,” Biophys. J. 100(8), L43–L45 (2011).
[Crossref] [PubMed]

Galbraith, C. G.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

Galbraith, J. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

Gan, Q.

M. Friedrich, Q. Gan, V. Ermolayev, and G. S. Harms, “STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution,” Biophys. J. 100(8), L43–L45 (2011).
[Crossref] [PubMed]

Gao, L.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

Gendron-Fitzpatrick, A.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

Giske, A.

Goodwin, P. M.

Greger, K.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Grigiene, J.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Gunn-Moore, F. J.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Gurchenkov, V.

Hagedorn, P. H.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Hammond, P.

E. Schimitschek, J. Trias, P. Hammond, and R. Atkins, “Laser performance and stability of fluorinated coumarin dyes,” Opt. Commun. 11(4), 352–355 (1974).
[Crossref]

Han, K. Y.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

Harke, B.

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

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Harms, G. S.

M. Friedrich, Q. Gan, V. Ermolayev, and G. S. Harms, “STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution,” Biophys. J. 100(8), L43–L45 (2011).
[Crossref] [PubMed]

Hein, B.

Hell, S. W.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

M. Reuss, J. Engelhardt, and S. W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation,” Opt. Express 18(2), 1049–1058 (2010).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[Crossref] [PubMed]

G. Moneron, R. Medda, B. Hein, A. Giske, V. Westphal, and S. W. Hell, “Fast STED microscopy with continuous wave fiber lasers,” Opt. Express 18(2), 1302–1309 (2010).
[Crossref] [PubMed]

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

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

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4pi fluorescence microscopy: theory and experiment,” New J. Phys. 7, 134 (2005).
[Crossref]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[Crossref]

M. Dyba and S. W. Hell, “Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[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 J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[Crossref] [PubMed]

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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.

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]

Johnson, M. L.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[Crossref] [PubMed]

Kasimova, M. R.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Kastrup, L.

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

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[Crossref]

Keller, J.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, 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. Lührmann, 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]

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4pi fluorescence microscopy: theory and experiment,” New J. Phys. 7, 134 (2005).
[Crossref]

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]

Krab, K.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[Crossref] [PubMed]

Leutenegger, M.

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

Marcello, M.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Medda, R.

G. Moneron, R. Medda, B. Hein, A. Giske, V. Westphal, and S. W. Hell, “Fast STED microscopy with continuous wave fiber lasers,” Opt. Express 18(2), 1302–1309 (2010).
[Crossref] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

Milkie, D. E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

Møller, I. M.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Moneron, G.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

G. Moneron, R. Medda, B. Hein, A. Giske, V. Westphal, and S. W. Hell, “Fast STED microscopy with continuous wave fiber lasers,” Opt. Express 18(2), 1302–1309 (2010).
[Crossref] [PubMed]

Munro, P. R. T.

Nassoy, P.

Nowaczyk, K.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[Crossref] [PubMed]

Nylk, J.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Pampaloni, F.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Planchon, T. A.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

Pollak, N.

N. Pollak, C. Dölle, and M. Ziegler, “The power to reduce: pyridine nucleotides - small molecules with a multitude of functions,” Biochem. J. 402(2), 205–218 (2007).
[Crossref] [PubMed]

Ramanujam, N.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

Reuss, M.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

M. Reuss, J. Engelhardt, and S. W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation,” Opt. Express 18(2), 1049–1058 (2010).
[Crossref] [PubMed]

Riching, K. M.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

Rittweger, E.

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

Rohrbach, A.

Schimitschek, E.

E. Schimitschek, J. Trias, P. Hammond, and R. Atkins, “Laser performance and stability of fluorinated coumarin dyes,” Opt. Commun. 11(4), 352–355 (1974).
[Crossref]

Schönle, A.

Skala, M. C.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

Stelzer, E. H.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Swoger, J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Szmacinski, H.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[Crossref] [PubMed]

Ta, H.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

Török, P.

Trias, J.

E. Schimitschek, J. Trias, P. Hammond, and R. Atkins, “Laser performance and stability of fluorinated coumarin dyes,” Opt. Commun. 11(4), 352–355 (1974).
[Crossref]

Ullal, C. K.

Verveer, P. J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Vettenburg, T.

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

Vicidomini, G.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

Werner, J. H.

Westphal, V.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

G. Moneron, R. Medda, B. Hein, A. Giske, V. Westphal, and S. W. Hell, “Fast STED microscopy with continuous wave fiber lasers,” Opt. Express 18(2), 1302–1309 (2010).
[Crossref] [PubMed]

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

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[Crossref]

White, J. G.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

Wichmann, J.

Wildanger, D.

Willig, K. I.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Zhang, P.

Ziegler, M.

N. Pollak, C. Dölle, and M. Ziegler, “The power to reduce: pyridine nucleotides - small molecules with a multitude of functions,” Biochem. J. 402(2), 205–218 (2007).
[Crossref] [PubMed]

Appl. Phys. Lett. (2)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[Crossref]

B. Boulanger, M. M. Fejer, R. Blachman, and P. F. Bordui, “Study of KTiOPO gray-tracking at 1064, 532, and 355 nm,” Appl. Phys. Lett. 65(19), 2401–2403 (1994).
[Crossref]

Biochem. J. (1)

N. Pollak, C. Dölle, and M. Ziegler, “The power to reduce: pyridine nucleotides - small molecules with a multitude of functions,” Biochem. J. 402(2), 205–218 (2007).
[Crossref] [PubMed]

Biophys. J. (1)

M. Friedrich, Q. Gan, V. Ermolayev, and G. S. Harms, “STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution,” Biophys. J. 100(8), L43–L45 (2011).
[Crossref] [PubMed]

Nat. Methods (6)

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).
[Crossref] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

T. Vettenburg, H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore, and K. Dholakia, “Light-sheet microscopy using an Airy beam,” Nat. Methods 11(5), 541–544 (2014).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

New J. Phys. (1)

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4pi fluorescence microscopy: theory and experiment,” New J. Phys. 7, 134 (2005).
[Crossref]

Opt. Commun. (1)

E. Schimitschek, J. Trias, P. Hammond, and R. Atkins, “Laser performance and stability of fluorinated coumarin dyes,” Opt. Commun. 11(4), 352–355 (1974).
[Crossref]

Opt. Express (8)

P. Török and P. R. T. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express 12(15), 3605–3617 (2004).
[Crossref] [PubMed]

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

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[Crossref] [PubMed]

M. Reuss, J. Engelhardt, and S. W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation,” Opt. Express 18(2), 1049–1058 (2010).
[Crossref] [PubMed]

P. Zhang, P. M. Goodwin, and J. H. Werner, “Fast, super resolution imaging via Bessel-beam stimulated emission depletion microscopy,” Opt. Express 22(10), 12398–12409 (2014).
[Crossref] [PubMed]

F. O. Fahrbach, V. Gurchenkov, K. Alessandri, P. Nassoy, and A. Rohrbach, “Self-reconstructing sectioned Bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy,” Opt. Express 21(9), 11425–11440 (2013).
[Crossref] [PubMed]

G. Moneron, R. Medda, B. Hein, A. Giske, V. Westphal, and S. W. Hell, “Fast STED microscopy with continuous wave fiber lasers,” Opt. Express 18(2), 1302–1309 (2010).
[Crossref] [PubMed]

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

Opt. Lett. (1)

Phys. Rev. Lett. (1)

M. Dyba and S. W. Hell, “Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[Crossref] [PubMed]

Plant Cell (1)

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (4)

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[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]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, 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]

Other (2)

L. Fulbert, J. Marty, B. Ferrand, and E. Molva, “Passively Q-switched monolithic microchip laser,” in Proc. of Conference on Laser and Electro Optics, vol. 15, paper CWC5 (Optical Society of America, Washington D.C., 1995), p. 176.

E. Cheng, R. Lane, J. W. L. Nighan, A. B. Petersen, J.-W. Pieterse, and C. Pohalski, “Q-switched laser system providing UV light,” US Patent # US 5835513 A (1998)

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Figures (8)

Fig. 1
Fig. 1

Normalized absorption and emission spectra of Coumarin 490 with laser wavelengths for excitation and stimulation.

Fig. 2
Fig. 2

Harmonic generation by a KTP-LBO tandem. The dotted red line represents the 1.06 μm beam, the green and the blue lines respectively the 532 and 355 nm beams. Crystals are embedded in hermetic boxes to protect them from humidity. For the SHG box, the front window is a half-wave plate at 1.06 μm to rotate the polarization to maximize the SHG efficiency on the KTP type II crystal, the back window is a half-wave plate at 1.06 μm and wave at 532 nm to make the polarizations of both wavelengths parallel and favor SFG conversion. The walk-off compensation is obtained by a 5 mm-thick silica slab approximately at Brewster incidence.

Fig. 3
Fig. 3

Experimental setup for measuring the STED SPIM efficiency of various dyes. The assembly of two waveplates at 532 and 355 nm with a broadband polarizer permits the modification of the power for the two colors independently. The upper part of the figure shows the detail of the beam shaping device with a typical beam shape on the far field of the 532 nm beam.

Fig. 4
Fig. 4

Images of the fluorescence trace when focusing into a solution of Coumarin 490 in ethanol with a 355 nm beam (top) and both 355 nm and 532 nm beams (bottom), at average powers of 1µw and 1mW respectively. The integrated profiles extracted from these images (center) show a strong fluorescence extinction at the focal point. The dip in the fluorescence near the waist of the 355 nm beam comes from a small bleaching of the dye which disappears by steering the solution.

Fig. 5
Fig. 5

Measurement of depletion efficiency in solution: (a) Normalized fluorescence profiles obtained in a Coumarin 490 solution in ethanol at different STED powers. The fit (solid black line) yields the value of γ according to the expression in the text. (b) Plot of the parameter γ obtained from the fits as a function of the STED power.

Fig. 6
Fig. 6

Images of the fluorescence trace in a spectrometer cell filled with a Coumarin 490 dye solution in ethanol, top with the 355 nm beam alone and bottom with donut shaped 532 nm beam added. The laser is focused by a F = 25 mm UV-Vis achromat with a numerical aperture NA~0.1. On the right: the profile plot of the images (with 1µW UV excitation alone in red and with 4 mW stimulation depletion in black) taken at distances of 40 µm (position a) and 100 µm (position b) from the UV beam waist.

Fig. 7
Fig. 7

Measurement of the fluorescent traces width under UV excitation alone (in black) and with green stimulation (in red) deduced from the two images shown in Fig. 6. The origin of the abscissa is taken at the waist of the UV-alone excited fluorescent trace. The vertical bars show the positions where the curves of Fig. 6 are taken.

Fig. 8
Fig. 8

Fluorescence traces section at the beam waist position as a function of the average STED power. The pump and the STED lasers where focussed by a 2.5 cm lens with a ~0.1 numerical aperture. The pump beam and the detections conditions stay constant as the STED beam varies. The graph on the right is a plot of the reciprocal of the squared FWHM of the fluorescence traces

Tables (1)

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Table 1 Experimental time-averaged saturation intensity for stimulation obtained with different compounds.

Equations (7)

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1 1+σ I STED τ
1 1+ I STED / I sat
w(z)= ( λ Z R π ( 1+ z 2 Z R 2 ) ) 1/2
I(z)= P λ Z R ( 1+ z 2 Z R 2 )
F(z) I exc (z)( 1 1+ I STED (z)/ I sat )
p(z)= F(z)dxdy P exc ( 1 1+ I STED (z) I sat )
p(z) ( 1+ P STED I sat λ Z R ( 1+ z 2 Z R 2 ) ) 1

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