Abstract

Two-photon scanning fluorescence microscopy has become a powerful tool for imaging living cells and tissues. Most applications of two-photon microscopy employ a Ti:sapphire laser excitation source, which is not readily portable or rapidly tunable. This work explores the use of two-photon fiber laser excitation (TP-FLEX) as an excitation source for scanning two-photon microscopy. We have further demonstrated the use of a photonic crystal fiber (PCF) for facile tuning of the excitation wavelength over the range from 810 nm to 1100 nm. We generated two-photon fluorescence images at excitation wavelengths from 850 nm to 1100 nm detected on a scanning-stage microscope. By PCF wavelength tuning the dye BODIPY fl was selectively excited at 1000 nm whereas MitoTracker red was excited preferentially at 1100 nm. We discuss the potential for fiber laser sources coupled with PCF wavelength tuning as an attractive tunable excitation source for two-photon scanning fluorescence microscopy.

© 2006 Optical Society of America

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  1. W. Denk, J. H. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990)
    [CrossRef] [PubMed]
  2. W. R. Zipfel, R. M. Williams and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat Biotechnol 21, 1369-1377 (2003).
    [CrossRef] [PubMed]
  3. A. Diaspro, Ed., Confocal and Two-Photon Microscopy. Foundations, Applications, and Advances, (Wiley-Liss, New York, 2002).
  4. K. König, "Multiphoton microscopy in life sciences," J Microsc. 200, 83-104 (2000).
    [CrossRef] [PubMed]
  5. C.-Y. Dong, K. H. Kim, C. Buehler, L. Hsu, H. Kim, P. T. C. So, B. R. Masters, E. Gratton and I. E. Kochevar, "Probing deep-tissue structures by two-photon fluorescence microscopy," Emerging Tools for Single-Cell Analysis 221-237 (2000).
    [CrossRef]
  6. J. C. Knight, T. A. Birks, P. S. J. Russell and D. M. Atkin, "All-Silica Single-Mode Optical Fiber with Photonic Crystal Cladding," Opt. Lett. 21, 1547-1549 (1996)
    [CrossRef] [PubMed]
  7. N. Nishizawa and T. Goto, "Widely wavelength-tunable ultrashort pulse generation using polarization maintaining optical fibers," IEEE J. Sel. Top. Quantum Electron. 7, 518-524 (2001).
    [CrossRef]
  8. M. A. Albota, C. Xu and W. W. Webb, "Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm," Appl. Opt. 37, 7352-7356 (1998).
    [CrossRef]
  9. G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
    [CrossRef]
  10. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
    [CrossRef] [PubMed]
  11. C. Xu and W. W. Webb, "Measurement of Two-Photon Excitation Cross Sections of Molecular Fluorophores with Data from 690 to 1050 nm," J. Opt. Soc. Am. B 13, 481-491 (1996).
    [CrossRef]
  12. G. P. Agrawal, Nonlinear Fiber Optics, (Academic, San Diego, CA,2001).
  13. J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
    [CrossRef]
  14. 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. 6,300-310 (2001).
    [CrossRef] [PubMed]
  15. M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
    [CrossRef] [PubMed]

2004 (1)

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

2003 (1)

W. R. Zipfel, R. M. Williams and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat Biotechnol 21, 1369-1377 (2003).
[CrossRef] [PubMed]

2001 (3)

N. Nishizawa and T. Goto, "Widely wavelength-tunable ultrashort pulse generation using polarization maintaining optical fibers," IEEE J. Sel. Top. Quantum Electron. 7, 518-524 (2001).
[CrossRef]

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef]

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. 6,300-310 (2001).
[CrossRef] [PubMed]

2000 (2)

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

K. König, "Multiphoton microscopy in life sciences," J Microsc. 200, 83-104 (2000).
[CrossRef] [PubMed]

1998 (1)

1996 (3)

J. C. Knight, T. A. Birks, P. S. J. Russell and D. M. Atkin, "All-Silica Single-Mode Optical Fiber with Photonic Crystal Cladding," Opt. Lett. 21, 1547-1549 (1996)
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
[CrossRef] [PubMed]

C. Xu and W. W. Webb, "Measurement of Two-Photon Excitation Cross Sections of Molecular Fluorophores with Data from 690 to 1050 nm," J. Opt. Soc. Am. B 13, 481-491 (1996).
[CrossRef]

1990 (1)

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

Albota, M. A.

Allen, M. W.

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

Arriaga, J.

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

Atkin, D. M.

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. 6,300-310 (2001).
[CrossRef] [PubMed]

Birks, T. A.

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

J. C. Knight, T. A. Birks, P. S. J. Russell and D. M. Atkin, "All-Silica Single-Mode Optical Fiber with Photonic Crystal Cladding," Opt. Lett. 21, 1547-1549 (1996)
[CrossRef] [PubMed]

Blab, G. A.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[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. 6,300-310 (2001).
[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. 6,300-310 (2001).
[CrossRef] [PubMed]

Cognet, L.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 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. 6,300-310 (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. 6,300-310 (2001).
[CrossRef] [PubMed]

Goto, T.

N. Nishizawa and T. Goto, "Widely wavelength-tunable ultrashort pulse generation using polarization maintaining optical fibers," IEEE J. Sel. Top. Quantum Electron. 7, 518-524 (2001).
[CrossRef]

Harms, G. S.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef]

Johnson, C. K.

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

Knight, J. C.

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

J. C. Knight, T. A. Birks, P. S. J. Russell and D. M. Atkin, "All-Silica Single-Mode Optical Fiber with Photonic Crystal Cladding," Opt. Lett. 21, 1547-1549 (1996)
[CrossRef] [PubMed]

König, K.

K. König, "Multiphoton microscopy in life sciences," J Microsc. 200, 83-104 (2000).
[CrossRef] [PubMed]

Lommerse, P. H. M.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef]

Nishizawa, N.

N. Nishizawa and T. Goto, "Widely wavelength-tunable ultrashort pulse generation using polarization maintaining optical fibers," IEEE J. Sel. Top. Quantum Electron. 7, 518-524 (2001).
[CrossRef]

Ortigosa-Blanch, A.

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

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. 6,300-310 (2001).
[CrossRef] [PubMed]

Russell, P. S.

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

Russell, P. S. J.

Schmidt, T.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef]

Shear, J. B.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
[CrossRef] [PubMed]

Strickler, J. H.

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

Urbauer, J. L.

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

Urbauer, R. J. B.

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

Wadsworth, W. J.

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

Webb, W. W.

W. R. Zipfel, R. M. Williams and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat Biotechnol 21, 1369-1377 (2003).
[CrossRef] [PubMed]

M. A. Albota, C. Xu and W. W. Webb, "Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm," Appl. Opt. 37, 7352-7356 (1998).
[CrossRef]

C. Xu and W. W. Webb, "Measurement of Two-Photon Excitation Cross Sections of Molecular Fluorophores with Data from 690 to 1050 nm," J. Opt. Soc. Am. B 13, 481-491 (1996).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
[CrossRef] [PubMed]

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

Williams, R. M.

W. R. Zipfel, R. M. Williams and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat Biotechnol 21, 1369-1377 (2003).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
[CrossRef] [PubMed]

Williams, T. D.

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

Xu, C.

M. A. Albota, C. Xu and W. W. Webb, "Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm," Appl. Opt. 37, 7352-7356 (1998).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
[CrossRef] [PubMed]

C. Xu and W. W. Webb, "Measurement of Two-Photon Excitation Cross Sections of Molecular Fluorophores with Data from 690 to 1050 nm," J. Opt. Soc. Am. B 13, 481-491 (1996).
[CrossRef]

Zaidi, A.

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

Zipfel, W.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat Biotechnol 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Anal. Biochem. (1)

M. W. Allen, R. J. B. Urbauer, A. Zaidi, T. D. Williams, J. L. Urbauer and C. K. Johnson, "Fluorescence Labeling, Purification and Immobilization of a Double Cysteine Mutant Calmodulin Fusion Protein for Single-Molecule Experiments," Anal. Biochem. 325,273-284 (2004).
[CrossRef] [PubMed]

Appl. Opt. (1)

Chem. Phys. Lett. (1)

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef]

IEEE J. Sel. Top. Quantum Electron. (1)

N. Nishizawa and T. Goto, "Widely wavelength-tunable ultrashort pulse generation using polarization maintaining optical fibers," IEEE J. Sel. Top. Quantum Electron. 7, 518-524 (2001).
[CrossRef]

IEEE Photon. Technol. Lett. (1)

J. C. Knight, J. Arriaga, T. A. Birks, A. Ortigosa-Blanch, W. J. Wadsworth and P. S. Russell, "Anomalous dispersion in photonic crystal fiber," IEEE Photon. Technol. Lett. 12, 807-809 (2000).
[CrossRef]

J Microsc. (1)

K. König, "Multiphoton microscopy in life sciences," J Microsc. 200, 83-104 (2000).
[CrossRef] [PubMed]

J. Biomed. Opt. (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. 6,300-310 (2001).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (1)

Nat Biotechnol (1)

W. R. Zipfel, R. M. Williams and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat Biotechnol 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Opt. Lett. (1)

Proceedings of the National Academy of Sciences of the United States of America (1)

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams and W. W. Webb, "Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy," Proceedings of the National Academy of Sciences of the United States of America 93, 10763-10768 (1996).
[CrossRef] [PubMed]

Science (1)

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

Other (3)

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

C.-Y. Dong, K. H. Kim, C. Buehler, L. Hsu, H. Kim, P. T. C. So, B. R. Masters, E. Gratton and I. E. Kochevar, "Probing deep-tissue structures by two-photon fluorescence microscopy," Emerging Tools for Single-Cell Analysis 221-237 (2000).
[CrossRef]

G. P. Agrawal, Nonlinear Fiber Optics, (Academic, San Diego, CA,2001).

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Figures (4)

Fig. 1.
Fig. 1.

Example of measured soliton wavelength shift by adjusting coupled power levels from a 20mW fiber laser into a 6 m PCF.

Fig. 2.
Fig. 2.

Two-photon fluorescence images of Fluocells® slide #1 with two-photon excitation wavelengths from 850 nm to 1100 nm. Average power at the back of the objective was approximately 800 µW at 1000 nm 800 µW at 1000 nm.

Fig. 3.
Fig. 3.

A. False color image combining the scans at 1100 nm (red) and 1000 nm (green) from Fig.2 showing selective excitation of F-actin filaments stained with Bodipy-Fl at 1000 nm and of mitochondria stained with MitoTracker Red at 1100 nm

Fig. 4.
Fig. 4.

Top: Relative two-photon excitation intensities for Bodipy-Fl and MitoTracker Red computed from areas where these species predominate in the images in Fig.2. Intensities were not corrected for the dependence of excitation power on wavelength (see Fig.1). Bottom: Ratio of fluorescence intensities of Bodipy-Fl intensity to Mitotracker Red over the range of excitation wavelengths shown in Fig.2. Lines are included merely to guide the eye.

Equations (1)

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2 π c γ P T 0 2 λ 2 D ( λ ) = 1 ,

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