Abstract

Saturated stimulated-emission depletion (STED) of a fluorescent marker has been shown to break the diffraction barrier in far-field fluorescence microscopy and to facilitate spatial resolution down to a few tens of nanometers. Here we investigate the photostability of a fluorophore that, in this concept, is repeatedly excited and depleted by synchronized laser pulses. Our study of bacteria labeled with RH-414, a membrane marker, reveals that increasing the duration of the STED pulse from ∼10 to 160 ps fundamentally improves the photostability of the dye. At the same time the STED efficiency is maintained. The observed photobleaching of RH-414 is due primarily to multiphoton absorption from its ground state. One can counteract photobleaching by employing STED pulses that range from 150 ps to approximately half of the lifetime of the excited state. The results also have implications for multiphoton excitation microscopy.

© 2003 Optical Society of America

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References

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  1. S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, eds. (Plenum, New York, 1997), pp. 361–426.
  2. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, S. W. Hell, “Fluorescence microscopy with diffraction resolution limit broken by stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
    [CrossRef]
  3. T. A. Klar, E. Engel, S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E 64, 066613 (2001).
    [CrossRef]
  4. M. Dyba, S. W. Hell, “Focal spots of size 1/23 open up far-field fluorescence microscopy at 33-nm axial resolution,” Phys. Rev. Lett. 88, 163901 (2002).
    [CrossRef]
  5. F. P. Schäfer, Dye Lasers (Springer-Verlag, Berlin, 1973).
  6. A. Grinvald, B. M. Salzberg, V. Lev-Ram, R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
    [CrossRef] [PubMed]
  7. V. Y. Artyukhov, E. I. Sinchenko, “Photoisomerization effect on fluorescence quenching in molecules containing a styryl group,” Russ. Phys. J. 44, 718–722 (2001).
    [CrossRef]
  8. P. E. Hänninen, M. Schrader, E. Soini, S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
    [CrossRef]
  9. H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
    [CrossRef] [PubMed]
  10. A. Hopt, E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
    [CrossRef] [PubMed]
  11. K. König, T. W. Becker, P. Fischer, I. Riemann, K.-J. Halbhuber, “Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes,” Opt. Lett. 24, 113–115 (1999).
    [CrossRef]

2002

M. Dyba, S. W. Hell, “Focal spots of size 1/23 open up far-field fluorescence microscopy at 33-nm axial resolution,” Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

2001

T. A. Klar, E. Engel, S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E 64, 066613 (2001).
[CrossRef]

V. Y. Artyukhov, E. I. Sinchenko, “Photoisomerization effect on fluorescence quenching in molecules containing a styryl group,” Russ. Phys. J. 44, 718–722 (2001).
[CrossRef]

A. Hopt, E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[CrossRef] [PubMed]

2000

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

1999

K. König, T. W. Becker, P. Fischer, I. Riemann, K.-J. Halbhuber, “Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes,” Opt. Lett. 24, 113–115 (1999).
[CrossRef]

H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
[CrossRef] [PubMed]

1995

P. E. Hänninen, M. Schrader, E. Soini, S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
[CrossRef]

1987

A. Grinvald, B. M. Salzberg, V. Lev-Ram, R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
[CrossRef] [PubMed]

Artyukhov, V. Y.

V. Y. Artyukhov, E. I. Sinchenko, “Photoisomerization effect on fluorescence quenching in molecules containing a styryl group,” Russ. Phys. J. 44, 718–722 (2001).
[CrossRef]

Baur, D.

H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
[CrossRef] [PubMed]

Becker, T. W.

Dyba, M.

M. Dyba, S. W. Hell, “Focal spots of size 1/23 open up far-field fluorescence microscopy at 33-nm axial resolution,” Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

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

Egner, A.

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

Engel, E.

T. A. Klar, E. Engel, S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E 64, 066613 (2001).
[CrossRef]

Fischer, P.

Grinvald, A.

A. Grinvald, B. M. Salzberg, V. Lev-Ram, R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
[CrossRef] [PubMed]

Halbhuber, K.-J.

Hänninen, P. E.

P. E. Hänninen, M. Schrader, E. Soini, S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
[CrossRef]

Hell, S. W.

M. Dyba, S. W. Hell, “Focal spots of size 1/23 open up far-field fluorescence microscopy at 33-nm axial resolution,” Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

T. A. Klar, E. Engel, S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E 64, 066613 (2001).
[CrossRef]

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

H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
[CrossRef] [PubMed]

P. E. Hänninen, M. Schrader, E. Soini, S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
[CrossRef]

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, eds. (Plenum, New York, 1997), pp. 361–426.

Hildesheim, R.

A. Grinvald, B. M. Salzberg, V. Lev-Ram, R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
[CrossRef] [PubMed]

Hopt, A.

A. Hopt, E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[CrossRef] [PubMed]

Jakobs, S.

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

Klar, T. A.

T. A. Klar, E. Engel, S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E 64, 066613 (2001).
[CrossRef]

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

Koester, H. J.

H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
[CrossRef] [PubMed]

König, K.

Lev-Ram, V.

A. Grinvald, B. M. Salzberg, V. Lev-Ram, R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
[CrossRef] [PubMed]

Neher, E.

A. Hopt, E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[CrossRef] [PubMed]

Riemann, I.

Salzberg, B. M.

A. Grinvald, B. M. Salzberg, V. Lev-Ram, R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
[CrossRef] [PubMed]

Schäfer, F. P.

F. P. Schäfer, Dye Lasers (Springer-Verlag, Berlin, 1973).

Schrader, M.

P. E. Hänninen, M. Schrader, E. Soini, S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
[CrossRef]

Sinchenko, E. I.

V. Y. Artyukhov, E. I. Sinchenko, “Photoisomerization effect on fluorescence quenching in molecules containing a styryl group,” Russ. Phys. J. 44, 718–722 (2001).
[CrossRef]

Soini, E.

P. E. Hänninen, M. Schrader, E. Soini, S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
[CrossRef]

Uhl, R.

H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
[CrossRef] [PubMed]

Bioimaging

P. E. Hänninen, M. Schrader, E. Soini, S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
[CrossRef]

Biophys. J.

H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
[CrossRef] [PubMed]

A. Hopt, E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[CrossRef] [PubMed]

A. Grinvald, B. M. Salzberg, V. Lev-Ram, R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
[CrossRef] [PubMed]

Opt. Lett.

Phys. Rev. E

T. A. Klar, E. Engel, S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E 64, 066613 (2001).
[CrossRef]

Phys. Rev. Lett.

M. Dyba, S. W. Hell, “Focal spots of size 1/23 open up far-field fluorescence microscopy at 33-nm axial resolution,” Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef]

Proc. Natl. Acad. Sci. USA

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

Russ. Phys. J.

V. Y. Artyukhov, E. I. Sinchenko, “Photoisomerization effect on fluorescence quenching in molecules containing a styryl group,” Russ. Phys. J. 44, 718–722 (2001).
[CrossRef]

Other

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, eds. (Plenum, New York, 1997), pp. 361–426.

F. P. Schäfer, Dye Lasers (Springer-Verlag, Berlin, 1973).

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Figures (5)

Fig. 1
Fig. 1

Excitation and fluorescence spectra of the membrane-incorporated styryl-pyridinium compound RH-414 (inset) (a), (b), (c) Photodamage models, both of which are consistent with the data. The photobleaching induced by the red beam has a quadratic or even higher-order intensity dependence and is almost certainly initiated from the ground state.

Fig. 2
Fig. 2

Confocal STED fluorescence microscope employing a Ti:sapphire laser that is synchronously pumping a frequency-doubling, optical parametric oscillator (OPO). Whereas the microscope produces the red STED pulses, the OPO renders the green excitation (Exc) pulses. The red pulses are power controlled by a liquid-crystal device (LPC), stretched by a pulse stretcher (PS) consisting of a double grating, and chopped (CH). The pulse timing is adjusted by an optical delay line. The green and the red pulses are coupled into the microscope via dichroic mirrors DC1 and DC2 and finally are focused through an objective lens into the marked bacterial membrane (inset). Fluorescence time curves are recorded by an avalanche photodiode that is synchronized with the chopper wheel. SHG, second-harmonic generator; PSFs, point-spread functions.

Fig. 3
Fig. 3

Raw time curves for configurations with (a) a leading and (b) a succeeding green excitation pulse, that is, with (a) regular and (b) reversed pulse time order. Each measurement consisted of three curves: I, green excitation only; II, green excitation with a chopped red beam; and IV, a chopped red beam only. Curve III represents the residual excitation caused by the red beam, that is, III = II - IV. Photodamage induced by the red beam is apparent in curves II and III. The annotation of the fluorescence intensity levels is used in calculating the efficiency of stimulated emission and photobleaching. The residual excitation through the red beam can reduce the STED efficiency at high peak intensities of the red beam. Here, curves are displayed for 13-ps red pulses at average powers of (a) 3.67 and (b) 3.51 mW.

Fig. 4
Fig. 4

Photobleaching by the red beam as a function of the pulse duration, studied for preceding red pulses. (a) Background-corrected time curves of type III recorded with pulses of durations τred = 13, 40, 85, 156 ps but at the same focal average power of 5.52 mW. The strong increase of photobleaching with reduced pulse duration indicates a nonlinear behavior of photobleaching. (b) Bleaching efficiency βrev as a function of average power of the red beam, P red. Whereas the shorter pulses lead to increased photobleaching at higher average power, for τred = 156 ps the bleaching efficiency is constant within the power range investigated.

Fig. 5
Fig. 5

Efficiency of stimulated emission and photobleaching for succeeding red pulses as a function of power P red and pulse duration τred. (a) ηgross reveals the fraction of excited molecules after the red-pulse action, (b) ηSTED indicates the depletion of the excited state by stimulated emission, and (c) shows the efficiency of bleaching. ηgross is decreased by residual multiphoton excitation by the red beam, which becomes stronger for decreasing τred and higher P red. In (b) it is shown that the depletion by stimulated emission does not depend on the pulse length. Therefore long pulses should be used for efficient depletion and low photobleaching.

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