Abstract

Most of the two-photon fluorescence microscopes are based on femtosecond Ti:Sapphire laser sources near the 800 nm wavelength. Here, we introduce a new confocal two-photon microscope system using a mode-locked Yb3+-doped fiber laser. The mode-locked fiber laser produces 13 ps pulses with large positive chirping at a repetition rate of 36 MHz with an average power of 80 mW. By using an external grating pair pulse compressor, the pulse width and the frequency chirping of the laser output are controlled for optimum two-photon excitation. For a given objective lens, the optimum condition was obtained by monitoring the two-photon-induced-photocurrent in a GaAsP photodiode at the sample position. The performance of this pulse width optimized two-photon microscope system was demonstrated by imaging Vybrant DiI-stained dorsal root ganglion cells in 2 and 3 dimensions.

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References

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [CrossRef] [PubMed]
  2. A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
    [CrossRef] [PubMed]
  3. M. Sridhar, S. Basu, V. L. Scranton, and P. J. Campagnola, “Construction of a laser scanning microscope for multiphoton excited optical fabrication,” Rev. Sci. Instrum. 74(7), 3474–3477 (2003).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  10. S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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2011 (1)

D. U. Kim, S. Moon, H. Song, H.-S. Kwon, and D. Y. Kim, “Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope,” Scanning 33(6), 455–462 (2011).
[CrossRef] [PubMed]

2009 (2)

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
[CrossRef] [PubMed]

J. J. Mancuso, A. M. Larson, T. G. Wensel, and P. Saggau, “Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging,” J. Biomed. Opt. 14(3), 034048 (2009).
[CrossRef] [PubMed]

2007 (2)

2006 (3)

2004 (1)

2003 (1)

M. Sridhar, S. Basu, V. L. Scranton, and P. J. Campagnola, “Construction of a laser scanning microscope for multiphoton excited optical fabrication,” Rev. Sci. Instrum. 74(7), 3474–3477 (2003).
[CrossRef]

2001 (1)

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

1997 (1)

1995 (1)

1991 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1988 (1)

P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988).
[CrossRef]

1987 (1)

1975 (1)

G. Koren, “Two-photon photoconductivity phenomena in semiconductors and insulators,” Phys. Rev. B 11(2), 802–821 (1975).
[CrossRef]

Andresen, V.

E. Büttner, V. Andresen, I. Rimke, and P. Friedl, “Infrared multiphoton microscopy beyond 1 micron: system design and biomedical applications,” Proc. SPIE6630, 66300H1– 66300H-8 (2007)

Bado, P.

P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988).
[CrossRef]

Baltuska, A.

Basu, S.

M. Sridhar, S. Basu, V. L. Scranton, and P. J. Campagnola, “Construction of a laser scanning microscope for multiphoton excited optical fabrication,” Rev. Sci. Instrum. 74(7), 3474–3477 (2003).
[CrossRef]

Becker, P. C.

Beretta, S.

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

Büttner, E.

E. Büttner, V. Andresen, I. Rimke, and P. Friedl, “Infrared multiphoton microscopy beyond 1 micron: system design and biomedical applications,” Proc. SPIE6630, 66300H1– 66300H-8 (2007)

Campagnola, P. J.

M. Sridhar, S. Basu, V. L. Scranton, and P. J. Campagnola, “Construction of a laser scanning microscope for multiphoton excited optical fabrication,” Rev. Sci. Instrum. 74(7), 3474–3477 (2003).
[CrossRef]

Cannone, F.

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

Chan, M. C.

Chen, L. J.

Chen, Z.

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
[CrossRef] [PubMed]

Chirico, G.

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

Chong, A.

Cruz, C. H.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Diaspro, A.

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

Federici, F.

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

Fermann, M. E.

Fork, R. L.

Friedl, P.

E. Büttner, V. Andresen, I. Rimke, and P. Friedl, “Infrared multiphoton microscopy beyond 1 micron: system design and biomedical applications,” Proc. SPIE6630, 66300H1– 66300H-8 (2007)

Gaeta, A. L.

Guo, H.

Guol, S. H.

Haberl, F.

Hashimoto, T.

Haus, H. A.

Hofer, M.

Hui, R.

J. R. Unruh, E. S. Price, R. G. Molla, L. Stehno-Bittel, C. K. Johnson, and R. Hui, “Two-photon microscopy with wavelength switchable fiber laser excitation,” Opt. Express 14(21), 9825–9831 (2006).
[CrossRef] [PubMed]

J. R. Unruh, E. S. Price, R. G. Molla, R. Hui, and C. K. Johnson, “Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy,” Microsc. Res. Tech. 69(11), 891–893 (2006).
[CrossRef] [PubMed]

Ippen, E. P.

Ito, H.

Johnson, C. K.

J. R. Unruh, E. S. Price, R. G. Molla, R. Hui, and C. K. Johnson, “Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy,” Microsc. Res. Tech. 69(11), 891–893 (2006).
[CrossRef] [PubMed]

J. R. Unruh, E. S. Price, R. G. Molla, L. Stehno-Bittel, C. K. Johnson, and R. Hui, “Two-photon microscopy with wavelength switchable fiber laser excitation,” Opt. Express 14(21), 9825–9831 (2006).
[CrossRef] [PubMed]

Kim, D. U.

D. U. Kim, S. Moon, H. Song, H.-S. Kwon, and D. Y. Kim, “Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope,” Scanning 33(6), 455–462 (2011).
[CrossRef] [PubMed]

Kim, D. Y.

D. U. Kim, S. Moon, H. Song, H.-S. Kwon, and D. Y. Kim, “Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope,” Scanning 33(6), 455–462 (2011).
[CrossRef] [PubMed]

Koren, G.

G. Koren, “Two-photon photoconductivity phenomena in semiconductors and insulators,” Phys. Rev. B 11(2), 802–821 (1975).
[CrossRef]

Krasieva, T. B.

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
[CrossRef] [PubMed]

Kwon, H.-S.

D. U. Kim, S. Moon, H. Song, H.-S. Kwon, and D. Y. Kim, “Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope,” Scanning 33(6), 455–462 (2011).
[CrossRef] [PubMed]

Larson, A. M.

J. J. Mancuso, A. M. Larson, T. G. Wensel, and P. Saggau, “Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging,” J. Biomed. Opt. 14(3), 034048 (2009).
[CrossRef] [PubMed]

Lenz, G.

Liu, J.

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
[CrossRef] [PubMed]

Maine, P.

P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988).
[CrossRef]

Mancuso, J. J.

J. J. Mancuso, A. M. Larson, T. G. Wensel, and P. Saggau, “Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging,” J. Biomed. Opt. 14(3), 034048 (2009).
[CrossRef] [PubMed]

Molla, R. G.

J. R. Unruh, E. S. Price, R. G. Molla, R. Hui, and C. K. Johnson, “Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy,” Microsc. Res. Tech. 69(11), 891–893 (2006).
[CrossRef] [PubMed]

J. R. Unruh, E. S. Price, R. G. Molla, L. Stehno-Bittel, C. K. Johnson, and R. Hui, “Two-photon microscopy with wavelength switchable fiber laser excitation,” Opt. Express 14(21), 9825–9831 (2006).
[CrossRef] [PubMed]

Moon, S.

D. U. Kim, S. Moon, H. Song, H.-S. Kwon, and D. Y. Kim, “Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope,” Scanning 33(6), 455–462 (2011).
[CrossRef] [PubMed]

Mourou, G.

P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988).
[CrossRef]

Ober, M. H.

Pessot, M.

P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988).
[CrossRef]

Price, E. S.

J. R. Unruh, E. S. Price, R. G. Molla, R. Hui, and C. K. Johnson, “Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy,” Microsc. Res. Tech. 69(11), 891–893 (2006).
[CrossRef] [PubMed]

J. R. Unruh, E. S. Price, R. G. Molla, L. Stehno-Bittel, C. K. Johnson, and R. Hui, “Two-photon microscopy with wavelength switchable fiber laser excitation,” Opt. Express 14(21), 9825–9831 (2006).
[CrossRef] [PubMed]

Pshenichnikov, M. S.

Ranka, J. K.

Renninger, W. H.

Rimke, I.

E. Büttner, V. Andresen, I. Rimke, and P. Friedl, “Infrared multiphoton microscopy beyond 1 micron: system design and biomedical applications,” Proc. SPIE6630, 66300H1– 66300H-8 (2007)

Robello, M.

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

Saggau, P.

J. J. Mancuso, A. M. Larson, T. G. Wensel, and P. Saggau, “Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging,” J. Biomed. Opt. 14(3), 034048 (2009).
[CrossRef] [PubMed]

Sato, K.

Schmidt, A. J.

Scranton, V. L.

M. Sridhar, S. Basu, V. L. Scranton, and P. J. Campagnola, “Construction of a laser scanning microscope for multiphoton excited optical fabrication,” Rev. Sci. Instrum. 74(7), 3474–3477 (2003).
[CrossRef]

Shank, C. V.

Song, H.

D. U. Kim, S. Moon, H. Song, H.-S. Kwon, and D. Y. Kim, “Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope,” Scanning 33(6), 455–462 (2011).
[CrossRef] [PubMed]

Sridhar, M.

M. Sridhar, S. Basu, V. L. Scranton, and P. J. Campagnola, “Construction of a laser scanning microscope for multiphoton excited optical fabrication,” Rev. Sci. Instrum. 74(7), 3474–3477 (2003).
[CrossRef]

Stehno-Bittel, L.

Strickland, D.

P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Sun, C. K.

Tai, S. P.

Taira, K.

Takashima, K.

Tamura, K.

Tang, S.

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
[CrossRef] [PubMed]

Taniguchi, H.

Tromberg, B. J.

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
[CrossRef] [PubMed]

Tsai, T. H.

Unruh, J. R.

J. R. Unruh, E. S. Price, R. G. Molla, R. Hui, and C. K. Johnson, “Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy,” Microsc. Res. Tech. 69(11), 891–893 (2006).
[CrossRef] [PubMed]

J. R. Unruh, E. S. Price, R. G. Molla, L. Stehno-Bittel, C. K. Johnson, and R. Hui, “Two-photon microscopy with wavelength switchable fiber laser excitation,” Opt. Express 14(21), 9825–9831 (2006).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Wensel, T. G.

J. J. Mancuso, A. M. Larson, T. G. Wensel, and P. Saggau, “Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging,” J. Biomed. Opt. 14(3), 034048 (2009).
[CrossRef] [PubMed]

Wiersma, D. A.

Wise, F. W.

Yoda, T.

Yokoyama, H.

IEEE J. Quantum Electron. (1)

P. Maine, D. Strickland, P. Bado, M. Pessot, and G. Mourou, “Generation of ultrahigh peak power pulses by chirped pulse amplification,” IEEE J. Quantum Electron. 24(2), 398–403 (1988).
[CrossRef]

J. Biomed. Opt. (3)

A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, “Two-photon microscopy and spectroscopy based on a compact confocal scanning head,” J. Biomed. Opt. 6(3), 300–310 (2001).
[CrossRef] [PubMed]

J. J. Mancuso, A. M. Larson, T. G. Wensel, and P. Saggau, “Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging,” J. Biomed. Opt. 14(3), 034048 (2009).
[CrossRef] [PubMed]

S. Tang, J. Liu, T. B. Krasieva, Z. Chen, and B. J. Tromberg, “Developing compact multiphoton systems using femtosecond fiber lasers,” J. Biomed. Opt. 14(3), 030508 (2009).
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

J. R. Unruh, E. S. Price, R. G. Molla, R. Hui, and C. K. Johnson, “Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy,” Microsc. Res. Tech. 69(11), 891–893 (2006).
[CrossRef] [PubMed]

Opt. Express (4)

Opt. Lett. (5)

Phys. Rev. B (1)

G. Koren, “Two-photon photoconductivity phenomena in semiconductors and insulators,” Phys. Rev. B 11(2), 802–821 (1975).
[CrossRef]

Rev. Sci. Instrum. (1)

M. Sridhar, S. Basu, V. L. Scranton, and P. J. Campagnola, “Construction of a laser scanning microscope for multiphoton excited optical fabrication,” Rev. Sci. Instrum. 74(7), 3474–3477 (2003).
[CrossRef]

Scanning (1)

D. U. Kim, S. Moon, H. Song, H.-S. Kwon, and D. Y. Kim, “Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope,” Scanning 33(6), 455–462 (2011).
[CrossRef] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (5)

A. Diaspro, Confocal and Two-photon Microscopy Foundations, Applications, and Advances (Wiely-Liss, 2002), Chap. 8.

E. Büttner, V. Andresen, I. Rimke, and P. Friedl, “Infrared multiphoton microscopy beyond 1 micron: system design and biomedical applications,” Proc. SPIE6630, 66300H1– 66300H-8 (2007)

http://www.microscopyu.com/articles/optics/cfintro.html .

J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006), Chap. 9.

F. Träger, Handbook of Lasers and Optics (Springer, 2007), Chap. 12.

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Figures (5)

Fig. 1
Fig. 1

Schematic diagrams of our custom built two-photon microscope system. (a) a passively mode-locked fiber ring laser producing 36 MHz pulses with an external Yb3+ doped fiber amplifier, (b) a variable double-pass grating-pair pulse compressor, (c) a confocal two-photon microscope with a point-scanning galvanometer system. LD: laser diode, WDM: wavelength division multiplexer, YDF: Yb3+ doped fiber, YDFA: Yb3+ doped fiber amplifier, PBS: polarization beam splitter, PMT: photomultiplier tube.

Fig. 2
Fig. 2

(a) Spectrum and (b) autocorrelation traces of our fiber laser source before and after pulse compression.

Fig. 3
Fig. 3

(a) Lateral and (b) axial resolution. The intensity profiles of measured fluorescence intensities were fitted with Gaussian functions to obtain the FWHMs of measured intensities to obtain the lateral and the axial resolutions.

Fig. 4
Fig. 4

(a) PD signal as a function of the excitation power for CW and pulse operation. The squares are for a spot size of 250 μm in CW excitation and the circles (triangles) are for a spot size of 250 μm (2.5 μm) in pulsed excitation. (b) Normalized PD signals as a function of the grating separation length. Solid lines are least-square fitting curves with quadratic functions.

Fig. 5
Fig. 5

(a) 3D two-photon image of Vybrant DiI-stained DRG cells. (b), (c), and (d) 2D two-photon images from the 135th xy-plane, 100th xz-plane, and 255th yz-plane, respectively.

Equations (2)

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mλ=d(sinα+sinβ)
GDD= 1 1 ( λ c d sinα ) 2 λ c 3 2π c 2 d 2 L sep cos 2 β(ω)

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