Abstract

Spectral and temporal characterization is a fundamental task when a tunable Ti:sapphire ultrafast laser system is operated for multiphoton microscopy applications. In the present paper simple procedures are reported that perform laser-peak-emission wavelength and bandwidth measurements without the need of any further instrumentation but a simple and inexpensive diffraction grating, by taking advantage of the confocal microscope imaging capabilities.

© 2004 Optical Society of America

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References

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  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
    [CrossRef]
  3. K. Konig, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
    [CrossRef] [PubMed]
  4. A. Diaspro, ed. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (Wiley-Liss, New York, 2002).
  5. P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope,” Bioimaging 2, 117–121 (1994).
    [CrossRef]
  6. G. J. Brakenhoff, M. Müller, J. Squier, “Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation,” J. Microsc. 179, 253–260 (1995).
    [CrossRef]
  7. M. Müller, J. Squier, G. J. Brakenhoff, “Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption,” Opt. Lett. 20, 1038–1040 (1995).
    [CrossRef] [PubMed]
  8. J. B. Guild, C. Xu, W. W. Webb, “Measurement of group delay dispersion of high numerical aperture lenses using two-photon excited fluorescence,” Appl. Opt. 36, 397–401 (1997).
    [CrossRef] [PubMed]
  9. M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
    [CrossRef] [PubMed]
  10. R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B 67, 87–94 (1998).
    [CrossRef]
  11. A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
    [CrossRef]
  12. F. Cannone, G. Chirico, G. Baldini, A. Diaspro, “Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,” J. Microsc. 210, 149–157 (2003).
    [CrossRef] [PubMed]
  13. A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
    [CrossRef] [PubMed]
  14. M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, 1980), p. 412.
  15. M. Abramowitz, M. W. Davidson, “Microscope Objectives-Nikon CFI60 200/60/25 Specification,” (2003), http://micro.magnet.fsu.edu/primer/anatomy/nikoninfinity.html .
  16. J. M. Lerner, A. Thevenon, The Optics of Spectroscopy, paragraph 2.12.1 (Jobin Yvon, Edison, N.J., 1988), http://www.jobinyvon.com/usadivisions/oos/index.htm .
  17. J. C. Diels, W. Rudolph, Ultrashort Laser Pulse Phenomena: Fundamentals, Techniques, and Applications on a Femtosecond Time Scale (Academic, San Diego, 1996), p. 7.

2003 (1)

F. Cannone, G. Chirico, G. Baldini, A. Diaspro, “Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,” J. Microsc. 210, 149–157 (2003).
[CrossRef] [PubMed]

2000 (1)

K. Konig, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[CrossRef] [PubMed]

1999 (2)

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
[CrossRef]

1998 (2)

M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
[CrossRef] [PubMed]

R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B 67, 87–94 (1998).
[CrossRef]

1997 (1)

1995 (2)

G. J. Brakenhoff, M. Müller, J. Squier, “Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation,” J. Microsc. 179, 253–260 (1995).
[CrossRef]

M. Müller, J. Squier, G. J. Brakenhoff, “Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption,” Opt. Lett. 20, 1038–1040 (1995).
[CrossRef] [PubMed]

1994 (1)

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Baldini, G.

F. Cannone, G. Chirico, G. Baldini, A. Diaspro, “Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,” J. Microsc. 210, 149–157 (2003).
[CrossRef] [PubMed]

Born, M.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, 1980), p. 412.

Brakenhoff, G. J.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
[CrossRef] [PubMed]

G. J. Brakenhoff, M. Müller, J. Squier, “Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation,” J. Microsc. 179, 253–260 (1995).
[CrossRef]

M. Müller, J. Squier, G. J. Brakenhoff, “Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption,” Opt. Lett. 20, 1038–1040 (1995).
[CrossRef] [PubMed]

Cannone, F.

F. Cannone, G. Chirico, G. Baldini, A. Diaspro, “Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,” J. Microsc. 210, 149–157 (2003).
[CrossRef] [PubMed]

Chirico, G.

F. Cannone, G. Chirico, G. Baldini, A. Diaspro, “Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,” J. Microsc. 210, 149–157 (2003).
[CrossRef] [PubMed]

Corosu, M.

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Diaspro, A.

F. Cannone, G. Chirico, G. Baldini, A. Diaspro, “Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,” J. Microsc. 210, 149–157 (2003).
[CrossRef] [PubMed]

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Diels, J. C.

J. C. Diels, W. Rudolph, Ultrashort Laser Pulse Phenomena: Fundamentals, Techniques, and Applications on a Femtosecond Time Scale (Academic, San Diego, 1996), p. 7.

Feurer, T.

R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B 67, 87–94 (1998).
[CrossRef]

Fittinghoff, D. N.

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
[CrossRef]

Gaeta, A. L.

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
[CrossRef]

Guild, J. B.

Hänninen, P. E.

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

Hell, S. W.

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

Konig, K.

K. Konig, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[CrossRef] [PubMed]

Millard, A. C.

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
[CrossRef]

Müller, M.

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
[CrossRef]

M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
[CrossRef] [PubMed]

M. Müller, J. Squier, G. J. Brakenhoff, “Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption,” Opt. Lett. 20, 1038–1040 (1995).
[CrossRef] [PubMed]

G. J. Brakenhoff, M. Müller, J. Squier, “Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation,” J. Microsc. 179, 253–260 (1995).
[CrossRef]

Piston, D. W.

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Ramoino, P.

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Robello, M.

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Rudolph, W.

J. C. Diels, W. Rudolph, Ultrashort Laser Pulse Phenomena: Fundamentals, Techniques, and Applications on a Femtosecond Time Scale (Academic, San Diego, 1996), p. 7.

Sauerbrey, R.

R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B 67, 87–94 (1998).
[CrossRef]

Simon, U.

R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B 67, 87–94 (1998).
[CrossRef]

M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
[CrossRef] [PubMed]

Squier, J.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
[CrossRef] [PubMed]

M. Müller, J. Squier, G. J. Brakenhoff, “Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption,” Opt. Lett. 20, 1038–1040 (1995).
[CrossRef] [PubMed]

G. J. Brakenhoff, M. Müller, J. Squier, “Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation,” J. Microsc. 179, 253–260 (1995).
[CrossRef]

Squier, J. A.

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Webb, W. W.

J. B. Guild, C. Xu, W. W. Webb, “Measurement of group delay dispersion of high numerical aperture lenses using two-photon excited fluorescence,” Appl. Opt. 36, 397–401 (1997).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

Wolf, E.

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, 1980), p. 412.

Wolleschensky, R.

M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
[CrossRef] [PubMed]

R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B 67, 87–94 (1998).
[CrossRef]

Xu, C.

Appl. Opt. (1)

Appl. Phys. B (1)

R. Wolleschensky, T. Feurer, R. Sauerbrey, U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B 67, 87–94 (1998).
[CrossRef]

Bioimaging (1)

P. E. Hänninen, S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope,” Bioimaging 2, 117–121 (1994).
[CrossRef]

J. Microsc. (5)

G. J. Brakenhoff, M. Müller, J. Squier, “Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation,” J. Microsc. 179, 253–260 (1995).
[CrossRef]

M. Müller, J. Squier, R. Wolleschensky, U. Simon, G. J. Brakenhoff, “Dispersion precompensation of 15 femtosecond optical pulses for high numerical aperture objectives,” J. Microsc. 191, 141–150 (1998).
[CrossRef] [PubMed]

K. Konig, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[CrossRef] [PubMed]

A. C. Millard, D. N. Fittinghoff, J. A. Squier, M. Müller, A. L. Gaeta, “Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy,” J. Microsc. 193, 179–181 (1999).
[CrossRef]

F. Cannone, G. Chirico, G. Baldini, A. Diaspro, “Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method,” J. Microsc. 210, 149–157 (2003).
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

A. Diaspro, M. Corosu, P. Ramoino, M. Robello, “Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture,” Microsc. Res. Tech. 47, 196–205 (1999).
[CrossRef] [PubMed]

Opt. Lett. (1)

Science (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Other (6)

W. Denk, D. W. Piston, W. W. Webb, “Two-photon molecular excitation in laser-scanning microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1995), pp. 445–458.
[CrossRef]

A. Diaspro, ed. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (Wiley-Liss, New York, 2002).

M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, 1980), p. 412.

M. Abramowitz, M. W. Davidson, “Microscope Objectives-Nikon CFI60 200/60/25 Specification,” (2003), http://micro.magnet.fsu.edu/primer/anatomy/nikoninfinity.html .

J. M. Lerner, A. Thevenon, The Optics of Spectroscopy, paragraph 2.12.1 (Jobin Yvon, Edison, N.J., 1988), http://www.jobinyvon.com/usadivisions/oos/index.htm .

J. C. Diels, W. Rudolph, Ultrashort Laser Pulse Phenomena: Fundamentals, Techniques, and Applications on a Femtosecond Time Scale (Academic, San Diego, 1996), p. 7.

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Figures (9)

Fig. 1
Fig. 1

(a) Standard layout of a Confocal Multiphoton Microscope; (b) Picture of the instrument set up in our laboratory. A Nikon PCM2000 CLSM, equipped with a Nikon TE2000-U inverted optical microscope, has been directly coupled with a Mira 900 F Ti:sapphire oscillator pumped by a Verdi V5 frequency-doubled Nd:YVO4 laser (Coherent, Inc.).

Fig. 2
Fig. 2

Basic setup for wavelength measurements. A grating is placed onto the microscope specimen stage. The absence of any objective allows the far-field diffraction pattern to be imaged by the CLSM.

Fig. 3
Fig. 3

Three scanning angles at which the Littrow condition is fulfilled for the +1, 0, -1 diffracted orders, respectively.

Fig. 4
Fig. 4

Experimental setup for bandwidth measurements. A blazed grating is used here and is again placed onto the microscope specimen stage. The grating normal is rotated of an angle α n with respect to the z axis to allow the central wavelength λ at the nth order to be diffracted back along the z axis. The diffraction angles at which Littrow condition is fulfilled at wavelengths λ and λ ± Δλ are shown.

Fig. 5
Fig. 5

Typical image of the far-field diffraction pattern as it appears on the CLSM monitor. A 19.7-mm-1 spatial frequency Ronchi ruling has been used, a Ti:sapphire emission wavelength of 740 nm has been tuned, and the smallest 20-μm diameter pinhole has been selected. With a suitable choice of the display software setting parameters, wavelength values can be directly read as distances between diffraction order pairs. The angular spread of the diffracted order spots is about 0.3 mrad FWHM, corresponding to a spectral broadening of ∼30 nm.

Fig. 6
Fig. 6

Profile plot along a line section passing through the centers of the diffraction spots. The left part of the diffraction pattern, formally corresponding to negative wavelengths, bears no additional information.

Fig. 7
Fig. 7

The entire Ti:sapphire emission tuning curve has been explored, and the differences between measured and nominal wavelength values are plotted. All data lay around zero within the experimental uncertainty.

Fig. 8
Fig. 8

Typical diffraction patterns when bandwidth measurements are carried out. The Ti:sapphire oscillator has again been tuned around 740 nm. A blazed diffraction grating with a spatial frequency of 600 lp/mm has been used at the second order. The grating normal has been tilted around the optical z axis by an angle of 460 mrad. Shown are a diffraction pattern when (a) a continuous wave emission is generated and (b), when mode locking is active.

Fig. 9
Fig. 9

Intensity profiles versus Δλ along a central line section, corresponding to Figs. 8(a) and (b), respectively. The cw narrow laser linewidth yields a diffraction pattern [curve (a)], which is the instrumental spread function. Its angular dimension corresponds, in the present setup, to a spectrum width of ∼0.4 nm FWHM. When the laser linewidth broadens, owing to pulsed emission [curve (b)], a bandwidth of 4.7 nm results.

Equations (6)

Equations on this page are rendered with MathJax. Learn more.

2 sin α=ngλ; β=0,
α0=0, β0=0, α±1=±gλ/2, β±1=0,
x=αfob+xc, y=βfob+yc.
λ=2x+1-x0/gfob=Δx/fob2/g.
Δλ=Δxfob4ng2-λ21/2, Δy=0.
cτB/λ20.315,

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