Abstract

We present the implementation of intensity-modulated laser diodes for applications in frequency-domain pump–probe fluorescence microscopy. Our technique, which is based on the stimulated-emission approach, uses two sinusoidally modulated laser diodes. One laser (635 nm) excites the chromophores under study, and the other laser (680 nm) is responsible for inducing stimulated emission from excited-state molecules. Both light sources are modulated in the 80-MHz range but with an offset of 5 kHz between them. The result of the interaction of the pump and the probe beams is that a cross-correlation fluorescence signal at 5 kHz is generated primarily at the focal volume. Microscope imaging at the cross-correlation signal results in images with high contrast, and time-resolved high-frequency information can be acquired without high-speed detection. A detailed experimental arrangement of our methodology is presented along with images acquired from a 4.0-µm-diameter fluorescent sphere and TOTO-3–labeled mouse STO cells. (TOTO-3 is a nucleic acid stain.) Our results demonstrate the feasibility of using sinusoidally modulated laser diodes for pump–probe imaging, creating the exciting possibility of high-contrast time-resolved imaging with low-cost laser-diode systems.

© 2001 Optical Society of America

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References

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  1. D. K. Evans, ed., Laser Applications in Physical Chemistry (Marcel Dekker, New York, 1989).
  2. G. R. Fleming, Chemical Applications of Ultrafast Spectroscopy (Oxford U. Press, New York, 1986).
  3. F. E. Lytle, R. M. Parish, W. T. Barnes, “An introduction to time-resolved pump–probe spectroscopy,” Appl. Spectrosc. 39, 444–451 (1985).
    [CrossRef]
  4. J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.
  5. L. Xu, C. Spielmann, F. Krausz, R. Szipocs, “Ultrabroadband ring oscillator for sub-10-fs pulse generation,” Opt. Lett. 21, 1259–1261 (1996).
    [CrossRef] [PubMed]
  6. I. P. Christov, V. Stoev, M. M. Murnane, H. C. Kapteyn, “Sub-10-fs operation of Kerr-lens mode-locked lasers,” Opt. Lett. 21, 1493–1495 (1996).
    [CrossRef] [PubMed]
  7. R. M. Hochstrasser, C. K. Johnson, “Biological processes studied by ultrafast laser techniques,” in Ultrashort Laser Pulses, W. Kaiser, ed. (Springer-Verlag, New York, 1988), pp. 357–417.
    [CrossRef]
  8. P. A. Elzinga, R. J. Kneisler, F. E. Lytle, G. B. King, N. M. Laurendeau, “Pump–probe method for fast analysis of visible spectral signatures utilizing asynchronous optical sampling,” Appl. Opt. 26, 4303–4309 (1987).
    [CrossRef] [PubMed]
  9. P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
    [CrossRef]
  10. J. R. Lakowicz, I. Gryczynski, V. Bogdanov, J. Kusba, “Light quenching and fluorescence depolarization of Rhodamine-B and applications of this phenomenon to biophysics,” J. Phys. Chem. 98, 334–342 (1994).
    [CrossRef]
  11. J. Kusba, V. Bogdanov, I. Gryczynski, J. R. Lakowicz, “Theory of light quenching: effects on fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67, 2024–2040 (1994).
    [CrossRef]
  12. C. Y. Dong, P. T. C. So, T. French, E. Gratton, “Fluorescence lifetime imaging by asynchronous pump–probe microscopy,” Biophys. J. 69, 2234–2242 (1995).
    [CrossRef] [PubMed]
  13. C. Y. Dong, P. T. C. So, C. Buehler, E. Gratton, “Spatial resolution in scanning pump–probe fluorescence microscopy,” Optik 106, 7–14 (1997).
  14. C. Buehler, C. Y. Dong, P. T. C. So, E. Gratton, “Time-resolved polarization imaging by pump–probe (stimulated emission) fluorescence microscopy,” Biophys. J. 79, 536–549 (2000).
    [CrossRef] [PubMed]
  15. M. Born, E. Wolf, Principles of Optics, 5th ed. (Pergamon, Oxford, UK, 1985).
  16. C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).
  17. M. Gu, C. J. R. Sheppard, “Effects of a finite-sized pinhole on 3D image formation in confocal two-photon fluorescence microscopy,” J. Mod. Opt. 40, 2009–2024 (1993).
    [CrossRef]
  18. I. J. Cox, C. J. R. Sheppard, T. Wilson, “Superresolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).
  19. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
    [CrossRef]
  20. R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, 5th ed., K. D. Larison, ed. (Molecular Probes, Eugene, Ore., 1989).

2000 (1)

C. Buehler, C. Y. Dong, P. T. C. So, E. Gratton, “Time-resolved polarization imaging by pump–probe (stimulated emission) fluorescence microscopy,” Biophys. J. 79, 536–549 (2000).
[CrossRef] [PubMed]

1997 (1)

C. Y. Dong, P. T. C. So, C. Buehler, E. Gratton, “Spatial resolution in scanning pump–probe fluorescence microscopy,” Optik 106, 7–14 (1997).

1996 (2)

1995 (1)

C. Y. Dong, P. T. C. So, T. French, E. Gratton, “Fluorescence lifetime imaging by asynchronous pump–probe microscopy,” Biophys. J. 69, 2234–2242 (1995).
[CrossRef] [PubMed]

1994 (2)

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, J. Kusba, “Light quenching and fluorescence depolarization of Rhodamine-B and applications of this phenomenon to biophysics,” J. Phys. Chem. 98, 334–342 (1994).
[CrossRef]

J. Kusba, V. Bogdanov, I. Gryczynski, J. R. Lakowicz, “Theory of light quenching: effects on fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67, 2024–2040 (1994).
[CrossRef]

1993 (1)

M. Gu, C. J. R. Sheppard, “Effects of a finite-sized pinhole on 3D image formation in confocal two-photon fluorescence microscopy,” J. Mod. Opt. 40, 2009–2024 (1993).
[CrossRef]

1990 (1)

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

1987 (2)

P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
[CrossRef]

P. A. Elzinga, R. J. Kneisler, F. E. Lytle, G. B. King, N. M. Laurendeau, “Pump–probe method for fast analysis of visible spectral signatures utilizing asynchronous optical sampling,” Appl. Opt. 26, 4303–4309 (1987).
[CrossRef] [PubMed]

1985 (1)

F. E. Lytle, R. M. Parish, W. T. Barnes, “An introduction to time-resolved pump–probe spectroscopy,” Appl. Spectrosc. 39, 444–451 (1985).
[CrossRef]

1982 (1)

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Superresolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

Barnes, W. T.

F. E. Lytle, R. M. Parish, W. T. Barnes, “An introduction to time-resolved pump–probe spectroscopy,” Appl. Spectrosc. 39, 444–451 (1985).
[CrossRef]

Bogdanov, V.

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, J. Kusba, “Light quenching and fluorescence depolarization of Rhodamine-B and applications of this phenomenon to biophysics,” J. Phys. Chem. 98, 334–342 (1994).
[CrossRef]

J. Kusba, V. Bogdanov, I. Gryczynski, J. R. Lakowicz, “Theory of light quenching: effects on fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67, 2024–2040 (1994).
[CrossRef]

Born, M.

M. Born, E. Wolf, Principles of Optics, 5th ed. (Pergamon, Oxford, UK, 1985).

Buehler, C.

C. Buehler, C. Y. Dong, P. T. C. So, E. Gratton, “Time-resolved polarization imaging by pump–probe (stimulated emission) fluorescence microscopy,” Biophys. J. 79, 536–549 (2000).
[CrossRef] [PubMed]

C. Y. Dong, P. T. C. So, C. Buehler, E. Gratton, “Spatial resolution in scanning pump–probe fluorescence microscopy,” Optik 106, 7–14 (1997).

Christov, I. P.

I. P. Christov, V. Stoev, M. M. Murnane, H. C. Kapteyn, “Sub-10-fs operation of Kerr-lens mode-locked lasers,” Opt. Lett. 21, 1493–1495 (1996).
[CrossRef] [PubMed]

J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.

Cox, I. J.

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Superresolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

Dong, C. Y.

C. Buehler, C. Y. Dong, P. T. C. So, E. Gratton, “Time-resolved polarization imaging by pump–probe (stimulated emission) fluorescence microscopy,” Biophys. J. 79, 536–549 (2000).
[CrossRef] [PubMed]

C. Y. Dong, P. T. C. So, C. Buehler, E. Gratton, “Spatial resolution in scanning pump–probe fluorescence microscopy,” Optik 106, 7–14 (1997).

C. Y. Dong, P. T. C. So, T. French, E. Gratton, “Fluorescence lifetime imaging by asynchronous pump–probe microscopy,” Biophys. J. 69, 2234–2242 (1995).
[CrossRef] [PubMed]

Elzinga, P. A.

P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
[CrossRef]

P. A. Elzinga, R. J. Kneisler, F. E. Lytle, G. B. King, N. M. Laurendeau, “Pump–probe method for fast analysis of visible spectral signatures utilizing asynchronous optical sampling,” Appl. Opt. 26, 4303–4309 (1987).
[CrossRef] [PubMed]

Fleming, G. R.

G. R. Fleming, Chemical Applications of Ultrafast Spectroscopy (Oxford U. Press, New York, 1986).

French, T.

C. Y. Dong, P. T. C. So, T. French, E. Gratton, “Fluorescence lifetime imaging by asynchronous pump–probe microscopy,” Biophys. J. 69, 2234–2242 (1995).
[CrossRef] [PubMed]

Gratton, E.

C. Buehler, C. Y. Dong, P. T. C. So, E. Gratton, “Time-resolved polarization imaging by pump–probe (stimulated emission) fluorescence microscopy,” Biophys. J. 79, 536–549 (2000).
[CrossRef] [PubMed]

C. Y. Dong, P. T. C. So, C. Buehler, E. Gratton, “Spatial resolution in scanning pump–probe fluorescence microscopy,” Optik 106, 7–14 (1997).

C. Y. Dong, P. T. C. So, T. French, E. Gratton, “Fluorescence lifetime imaging by asynchronous pump–probe microscopy,” Biophys. J. 69, 2234–2242 (1995).
[CrossRef] [PubMed]

Gryczynski, I.

J. Kusba, V. Bogdanov, I. Gryczynski, J. R. Lakowicz, “Theory of light quenching: effects on fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67, 2024–2040 (1994).
[CrossRef]

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, J. Kusba, “Light quenching and fluorescence depolarization of Rhodamine-B and applications of this phenomenon to biophysics,” J. Phys. Chem. 98, 334–342 (1994).
[CrossRef]

Gu, M.

M. Gu, C. J. R. Sheppard, “Effects of a finite-sized pinhole on 3D image formation in confocal two-photon fluorescence microscopy,” J. Mod. Opt. 40, 2009–2024 (1993).
[CrossRef]

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

Haugland, R. P.

R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, 5th ed., K. D. Larison, ed. (Molecular Probes, Eugene, Ore., 1989).

Hochstrasser, R. M.

R. M. Hochstrasser, C. K. Johnson, “Biological processes studied by ultrafast laser techniques,” in Ultrashort Laser Pulses, W. Kaiser, ed. (Springer-Verlag, New York, 1988), pp. 357–417.
[CrossRef]

Huang, C. P.

J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.

Jian, Y.

P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
[CrossRef]

Johnson, C. K.

R. M. Hochstrasser, C. K. Johnson, “Biological processes studied by ultrafast laser techniques,” in Ultrashort Laser Pulses, W. Kaiser, ed. (Springer-Verlag, New York, 1988), pp. 357–417.
[CrossRef]

Kapteyn, H. C.

I. P. Christov, V. Stoev, M. M. Murnane, H. C. Kapteyn, “Sub-10-fs operation of Kerr-lens mode-locked lasers,” Opt. Lett. 21, 1493–1495 (1996).
[CrossRef] [PubMed]

J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.

King, G. B.

P. A. Elzinga, R. J. Kneisler, F. E. Lytle, G. B. King, N. M. Laurendeau, “Pump–probe method for fast analysis of visible spectral signatures utilizing asynchronous optical sampling,” Appl. Opt. 26, 4303–4309 (1987).
[CrossRef] [PubMed]

P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
[CrossRef]

Kneisler, R. J.

Krausz, F.

Kusba, J.

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, J. Kusba, “Light quenching and fluorescence depolarization of Rhodamine-B and applications of this phenomenon to biophysics,” J. Phys. Chem. 98, 334–342 (1994).
[CrossRef]

J. Kusba, V. Bogdanov, I. Gryczynski, J. R. Lakowicz, “Theory of light quenching: effects on fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67, 2024–2040 (1994).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, J. Kusba, “Light quenching and fluorescence depolarization of Rhodamine-B and applications of this phenomenon to biophysics,” J. Phys. Chem. 98, 334–342 (1994).
[CrossRef]

J. Kusba, V. Bogdanov, I. Gryczynski, J. R. Lakowicz, “Theory of light quenching: effects on fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67, 2024–2040 (1994).
[CrossRef]

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
[CrossRef]

Laurendeau, N. M.

P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
[CrossRef]

P. A. Elzinga, R. J. Kneisler, F. E. Lytle, G. B. King, N. M. Laurendeau, “Pump–probe method for fast analysis of visible spectral signatures utilizing asynchronous optical sampling,” Appl. Opt. 26, 4303–4309 (1987).
[CrossRef] [PubMed]

Lytle, F. E.

P. A. Elzinga, R. J. Kneisler, F. E. Lytle, G. B. King, N. M. Laurendeau, “Pump–probe method for fast analysis of visible spectral signatures utilizing asynchronous optical sampling,” Appl. Opt. 26, 4303–4309 (1987).
[CrossRef] [PubMed]

P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
[CrossRef]

F. E. Lytle, R. M. Parish, W. T. Barnes, “An introduction to time-resolved pump–probe spectroscopy,” Appl. Spectrosc. 39, 444–451 (1985).
[CrossRef]

Murnane, M. M.

Murnane, N. M.

J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.

Parish, R. M.

F. E. Lytle, R. M. Parish, W. T. Barnes, “An introduction to time-resolved pump–probe spectroscopy,” Appl. Spectrosc. 39, 444–451 (1985).
[CrossRef]

Sheppard, C. J. R.

M. Gu, C. J. R. Sheppard, “Effects of a finite-sized pinhole on 3D image formation in confocal two-photon fluorescence microscopy,” J. Mod. Opt. 40, 2009–2024 (1993).
[CrossRef]

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Superresolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

So, P. T. C.

C. Buehler, C. Y. Dong, P. T. C. So, E. Gratton, “Time-resolved polarization imaging by pump–probe (stimulated emission) fluorescence microscopy,” Biophys. J. 79, 536–549 (2000).
[CrossRef] [PubMed]

C. Y. Dong, P. T. C. So, C. Buehler, E. Gratton, “Spatial resolution in scanning pump–probe fluorescence microscopy,” Optik 106, 7–14 (1997).

C. Y. Dong, P. T. C. So, T. French, E. Gratton, “Fluorescence lifetime imaging by asynchronous pump–probe microscopy,” Biophys. J. 69, 2234–2242 (1995).
[CrossRef] [PubMed]

Spielmann, C.

Stoev, V.

Szipocs, R.

Taft, G.

J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.

Wilson, T.

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Superresolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

Wolf, E.

M. Born, E. Wolf, Principles of Optics, 5th ed. (Pergamon, Oxford, UK, 1985).

Xu, L.

Zhou, J.

J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.

Appl. Spectrosc. (2)

F. E. Lytle, R. M. Parish, W. T. Barnes, “An introduction to time-resolved pump–probe spectroscopy,” Appl. Spectrosc. 39, 444–451 (1985).
[CrossRef]

P. A. Elzinga, F. E. Lytle, Y. Jian, G. B. King, N. M. Laurendeau, “Pump–probe spectroscopy by asynchronous optical sampling,” Appl. Spectrosc. 41, 2–4 (1987).
[CrossRef]

Appl. Opt. (1)

Biophys. J. (3)

J. Kusba, V. Bogdanov, I. Gryczynski, J. R. Lakowicz, “Theory of light quenching: effects on fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67, 2024–2040 (1994).
[CrossRef]

C. Y. Dong, P. T. C. So, T. French, E. Gratton, “Fluorescence lifetime imaging by asynchronous pump–probe microscopy,” Biophys. J. 69, 2234–2242 (1995).
[CrossRef] [PubMed]

C. Buehler, C. Y. Dong, P. T. C. So, E. Gratton, “Time-resolved polarization imaging by pump–probe (stimulated emission) fluorescence microscopy,” Biophys. J. 79, 536–549 (2000).
[CrossRef] [PubMed]

J. Mod. Opt. (1)

M. Gu, C. J. R. Sheppard, “Effects of a finite-sized pinhole on 3D image formation in confocal two-photon fluorescence microscopy,” J. Mod. Opt. 40, 2009–2024 (1993).
[CrossRef]

J. Phys. Chem. (1)

J. R. Lakowicz, I. Gryczynski, V. Bogdanov, J. Kusba, “Light quenching and fluorescence depolarization of Rhodamine-B and applications of this phenomenon to biophysics,” J. Phys. Chem. 98, 334–342 (1994).
[CrossRef]

Opt. Lett. (2)

Optik (3)

I. J. Cox, C. J. R. Sheppard, T. Wilson, “Superresolution by confocal fluorescent microscopy,” Optik 60, 391–396 (1982).

C. Y. Dong, P. T. C. So, C. Buehler, E. Gratton, “Spatial resolution in scanning pump–probe fluorescence microscopy,” Optik 106, 7–14 (1997).

C. J. R. Sheppard, M. Gu, “Image formation in two-photon fluorescence microscopy,” Optik 86, 104–106 (1990).

Other (7)

M. Born, E. Wolf, Principles of Optics, 5th ed. (Pergamon, Oxford, UK, 1985).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
[CrossRef]

R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, 5th ed., K. D. Larison, ed. (Molecular Probes, Eugene, Ore., 1989).

R. M. Hochstrasser, C. K. Johnson, “Biological processes studied by ultrafast laser techniques,” in Ultrashort Laser Pulses, W. Kaiser, ed. (Springer-Verlag, New York, 1988), pp. 357–417.
[CrossRef]

J. Zhou, G. Taft, C. P. Huang, I. P. Christov, H. C. Kapteyn, N. M. Murnane, “Sub-10 fs pulse generation in Ti:sapphire: capabilities and ultimate limits,” in Ultrafast Phenomena IX, P. F. Barbara, W. H. Knox, G. A. Mourou, A. H. Zewail, eds. (Springer-Verlag, Berlin, 1995), pp. 39–40.

D. K. Evans, ed., Laser Applications in Physical Chemistry (Marcel Dekker, New York, 1989).

G. R. Fleming, Chemical Applications of Ultrafast Spectroscopy (Oxford U. Press, New York, 1986).

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Figures (4)

Fig. 1
Fig. 1

Frequency-domain pump–probe technique based on the principles of stimulated emission. stim.em, stimulated emission.

Fig. 2
Fig. 2

Design of a pump–probe fluorescence microscope that uses intensity-modulated laser diodes. D1 and D2, dichroic mirrors; LD, laser diode; PD, photodetector; ND, neutral-density filter; PZT DRV, piezoelectric driver; A, amplifier.

Fig. 3
Fig. 3

Pump–probe time-resolved image of a Crimson FluoSphere (4.0-µm diameter): (a) The harmonic amplitude of the cross-correlated signal. (b) The lifetime phase of the sphere at 80 MHz (τ = 3.59 ns). (c) Phase image of the reference compound Nile Blue. (d) Phase histogram of the sphere; the y axis represents the relative contribution of the plotted phase value.

Fig. 4
Fig. 4

Pump–probe time-resolved image of mouse STO cells that are labeled with the nucleic acid stain TOTO-3: (a) The harmonic amplitude of the cross-correlated signal. (b) The lifetime phase of the labeled nuclei at 80 MHz [τ = 1.81 ns (estimated)]. (c) Phase histogram of the labeled cells; the y axis represents the relative contribution of the plotted phase value.

Equations (5)

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d N r ,   t d t = - 1 τ   N r ,   t + σ λ I r ,   t c - N r ,   t - σ λ I r ,   t N r ,   t ,
F cc t = qc τ σ λ σ λ 2 1 1 + ω 2 1 / 2 cos ω - ω t - ϕ × I r I r d 3 r .
I r I r d 3 r .
I u ,   v I u ,   v ,
I u ,   v = 2 0 1 J 0 v ρ exp - iu ρ 2 / 2 ρ d ρ 2 ,

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