Abstract

We present a compact second-harmonic-generation (SHG) microscope based on a three-port single-mode fiber coupler. The fiber coupler is used to deliver a near-infrared ultrashort-pulsed laser beam as well as to collect the SHG signal in the visible wavelength range. The SHG microscope exhibits an axial resolution of 1.8 µm, which shows a slight enhancement of the optical sectioning effect compared with that for two-photon excitation at the same excitation wavelength. It is also demonstrated that SHG and two-photon fluorescence images under parallel and perpendicular laser excitation polarization can be simultaneously obtained.

© 2005 Optical Society of America

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  1. Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, Opt. Lett. 22, 1323 (1997).
    [CrossRef]
  2. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
    [CrossRef]
  3. A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. USA 99, 11,014 (2002).
    [CrossRef]
  4. D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
    [CrossRef]
  5. W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
    [CrossRef]
  6. P. J. Campagnola and L. M. Loew, Nat. Biotechnol. 21, 1356 (2003).
    [CrossRef] [PubMed]
  7. D. Bird and M. Gu, Opt. Lett. 27, 1031 (2002).
    [CrossRef]
  8. D. Bird and M. Gu, Opt. Lett. 28, 1552 (2003).
    [CrossRef] [PubMed]
  9. D. Bird and M. Gu, Appl. Opt. 41, 1852 (2002).
    [CrossRef] [PubMed]
  10. D. Bird and M. Gu, J. Microsc. (Oxford) 208, 35 (2002).
    [CrossRef]
  11. G. P. Agrawal, Nonlinear Fiber Optics (Academic, San Diego, Calif., 1989).
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    [CrossRef] [PubMed]
  13. L. Liu, X. Deng, L. Yang, G. Wang, and Z. Xu, Opt. Lett. 25, 1711 (2000).
    [CrossRef]
  14. L. Fu, X. Gan, D. Bird, and M. Gu, “Polarisation characteristics of a 1 × 2 fibre coupler under femtosecond pulsed and continuous wave illumination,” Opt. Laser Technol. (to be published).

2003 (4)

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

P. J. Campagnola and L. M. Loew, Nat. Biotechnol. 21, 1356 (2003).
[CrossRef] [PubMed]

D. Bird and M. Gu, Opt. Lett. 28, 1552 (2003).
[CrossRef] [PubMed]

2002 (5)

D. Bird and M. Gu, J. Microsc. (Oxford) 208, 35 (2002).
[CrossRef]

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. USA 99, 11,014 (2002).
[CrossRef]

D. Bird and M. Gu, Appl. Opt. 41, 1852 (2002).
[CrossRef] [PubMed]

D. Bird and M. Gu, Opt. Lett. 27, 1031 (2002).
[CrossRef]

2000 (1)

1999 (1)

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).
[CrossRef] [PubMed]

1997 (1)

Agrawal, G. P.

G. P. Agrawal, Nonlinear Fiber Optics (Academic, San Diego, Calif., 1989).

Alfano, R. R.

Bird, D.

D. Bird and M. Gu, Opt. Lett. 28, 1552 (2003).
[CrossRef] [PubMed]

D. Bird and M. Gu, Opt. Lett. 27, 1031 (2002).
[CrossRef]

D. Bird and M. Gu, J. Microsc. (Oxford) 208, 35 (2002).
[CrossRef]

D. Bird and M. Gu, Appl. Opt. 41, 1852 (2002).
[CrossRef] [PubMed]

L. Fu, X. Gan, D. Bird, and M. Gu, “Polarisation characteristics of a 1 × 2 fibre coupler under femtosecond pulsed and continuous wave illumination,” Opt. Laser Technol. (to be published).

Campagnola, P. J.

P. J. Campagnola and L. M. Loew, Nat. Biotechnol. 21, 1356 (2003).
[CrossRef] [PubMed]

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).
[CrossRef] [PubMed]

Christie, R.

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Deng, X.

Dombeck, D. A.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

Fu, L.

L. Fu, X. Gan, D. Bird, and M. Gu, “Polarisation characteristics of a 1 × 2 fibre coupler under femtosecond pulsed and continuous wave illumination,” Opt. Laser Technol. (to be published).

Gan, X.

L. Fu, X. Gan, D. Bird, and M. Gu, “Polarisation characteristics of a 1 × 2 fibre coupler under femtosecond pulsed and continuous wave illumination,” Opt. Laser Technol. (to be published).

Gu, M.

D. Bird and M. Gu, Opt. Lett. 28, 1552 (2003).
[CrossRef] [PubMed]

D. Bird and M. Gu, Opt. Lett. 27, 1031 (2002).
[CrossRef]

D. Bird and M. Gu, J. Microsc. (Oxford) 208, 35 (2002).
[CrossRef]

D. Bird and M. Gu, Appl. Opt. 41, 1852 (2002).
[CrossRef] [PubMed]

L. Fu, X. Gan, D. Bird, and M. Gu, “Polarisation characteristics of a 1 × 2 fibre coupler under femtosecond pulsed and continuous wave illumination,” Opt. Laser Technol. (to be published).

Guo, Y.

Harris, D.

Ho, P. P.

Hoppe, P. E.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Hyman, B. T.

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

Ingelsson, M.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

Kasischke, K. A.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

Lewis, A.

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).
[CrossRef] [PubMed]

Liu, F.

Liu, L.

Loew, L. M.

P. J. Campagnola and L. M. Loew, Nat. Biotechnol. 21, 1356 (2003).
[CrossRef] [PubMed]

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).
[CrossRef] [PubMed]

Malone, C. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Millard, A. C.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Mohler, W. A.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Nikitin, A. Y.

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Sacks, P.

Savage, H.

Schantz, S.

Terasaki, M.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Tromberg, B. J.

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. USA 99, 11,014 (2002).
[CrossRef]

Vishwasrao, H. D.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

Wang, G.

Webb, W. W.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Wei, M.

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).
[CrossRef] [PubMed]

Williams, R. M.

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Xu, Z.

Yang, L.

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. USA 99, 11,014 (2002).
[CrossRef]

Zhadin, N.

Zipfel, W. E.

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Zoumi, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. USA 99, 11,014 (2002).
[CrossRef]

Appl. Opt. (1)

Biophys. J. (2)

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

P. J. Campagnola, M. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (1999).
[CrossRef] [PubMed]

J. Microsc. (Oxford) (1)

D. Bird and M. Gu, J. Microsc. (Oxford) 208, 35 (2002).
[CrossRef]

Nat. Biotechnol. (1)

P. J. Campagnola and L. M. Loew, Nat. Biotechnol. 21, 1356 (2003).
[CrossRef] [PubMed]

Opt. Lett. (4)

Proc. Natl. Acad. Sci. USA (3)

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. USA 99, 11,014 (2002).
[CrossRef]

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7081 (2003).
[CrossRef]

W. E. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Other (2)

G. P. Agrawal, Nonlinear Fiber Optics (Academic, San Diego, Calif., 1989).

L. Fu, X. Gan, D. Bird, and M. Gu, “Polarisation characteristics of a 1 × 2 fibre coupler under femtosecond pulsed and continuous wave illumination,” Opt. Laser Technol. (to be published).

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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of the SHG microscope with a single-mode fiber coupler: (a) coupling efficiency from ports 2 and 3 to port 1; (b)–(d) mode profiles in the excitation and signal arms when a visible beam is coupled to port 1. ND, neutral density.

Fig. 2
Fig. 2

Emission spectra of AF-50 dye at 800 nm. Inset, SHG peak intensity as a function of excitation wavelength. The solid curve in the inset is derived from the spectra collected by focusing the output beam from port 2 of the fiber coupler into the spectrograph; the dashed curve is derived from the spectra collected directly from the sample without use of the fiber coupler. The excitation power is approximately 4 mW on the sample, and the excitation polarization is parallel to the fundamental laser polarization. TPEF, two-photon-excited fluorescence.

Fig. 3
Fig. 3

Quadratic dependence of SHG peak intensity on excitation power: (a) FWHM of axial responses as a function of the SHG wavelength and (b) axial response of the system for SHG signal and two-photon-excited (TPE) fluorescence from a thin layer of AF-50 dye. The excitation wavelength is 800 nm.

Fig. 4
Fig. 4

SHG and two-photon fluorescence images of a triangle-shaped paper sheet excited by parallel polarization (top) and perpendicular polarization (bottom) beams at an 800-nm wavelength. The excitation power is approximately 4 mW on the sample. SHG images (a) and (d) were obtained with a 400/9-nm bandpass filter; two-photon fluorescence images (b) and (e), with a 510/20-nm bandpass filter. Images in (e) and (f) were obtained by overlaying the SHG and two-photon fluorescence image. Scale bar, 50 µm.

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