We report on a novel and simple light source for short-wavelength two-photon excitation fluorescence microscopy based on the visible nonsolitonic radiation from a photonic crystal fiber. We demonstrate tunability of the light source by varying the wavelength and intensity of the Ti:Sapphire excitation light source. The visible nonsolitonic radiation is used as an excitation light source for two-photon fluorescence microscopy of tryptophan powder.

© 2005 Optical Society of America

1. G. P. Agrawal, Nonlinear fiber optics, 3rd ed. (Academic Press, San Diego, 2001).
2. J. Herrmann, U. Griebner, N. Zhavoronkov, A. Husakou, D. Nickel, J. C. Knight, W. J. Wadsworth, P. S. Russell, and G. Korn, "Experimental evidence for supercontinuum generation by fission of higher-order solitons in photonic fibers," Phys. Rev. Lett. 88, 173901 (2002); [CrossRef] [PubMed]
3. J. M. Dudley, L. Provino, N. Grossard, H. Maillotte, R. S. Windeler, B. J. Eggleton, and S. Coen, "Supercontinuum generation in air-silica microstructured fibers with nanosecond and femtosecond pulse pumping," J. Opt. Soc. Am. B 19, 765-771 (2002); [CrossRef]
4. A. V. Husakou and J. Herrmann, "Supercontinuum generation, four-wave mixing, and fission of higher-order solitons in photonic-crystal fibers," J. Opt. Soc. Am. B 19, 2171-2182 (2002); [CrossRef]
5. W. J. Wadsworth, A. Ortigosa-Blanch, J. C. Knight, T. A. Birks, T. P. M. Man, and P. S. Russell, "Supercontinuum generation in photonic crystal fibers and optical fiber tapers: a novel light source," J. Opt. Soc. Am. B 19, 2148-2155 (2002); [CrossRef]
6. J. K. Ranka, R. S. Windeler, and A. J. Stentz, "Optical properties of high-delta air-silica microstructure optical fibers," Opt. Lett. 25, 796-798 (2000); [CrossRef]
7. H. N. Paulsen, K. M. Hilligsoe, J. Thogersen, S. R. Keiding, and J. J. Larsen, "Coherent anti-Stokes Raman scattering microscopy with a photonic crystal fiber based light source," Opt. Lett. 28, 1123-1125 (2003); [CrossRef] [PubMed]
8. G. McConnell, "Confocal laser scanning fluorescence microscopy with a visible continuum source," Opt. Express 12, 2844-2850 (2004); [CrossRef] [PubMed]
9. C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo -Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, "An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy," J. Phys. D 37, 3296 (2004); [CrossRef]
10. G. McConnell and E. Riis, "Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2," Phys. Med. Biol. 49, 4757-4763 (2004); [CrossRef] [PubMed]
11. K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, "Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source," Jpn. J. Appl. Phys. Part 2 44, L167-L169 (2005); [CrossRef]
12. D. W. Piston, B. R. Masters, and W. W. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Microsc. 178 ( Pt 1), 20-27 (1995); [CrossRef]
13. W. R. Zipfel, R. M. Williams, R. Christie, A. Yu Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci. USA 100, 7075-7080 (2003); [CrossRef] [PubMed]
14. A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002); [CrossRef] [PubMed]
15. "Photonic Crystal Fiber Data Sheet (NL-1.5-670)" (Crystal Fibre A/S), retrieved April, 2005, <a href="http://www.crystal-fibre.com/datasheets/NL-15-670.pdf">http://www.crystal-fibre.com/datasheets/NL-15-670.pdf</a>
16. J. N. Elgin, T. Brabec, and S. M. J. Kelly, "A Perturbative Theory of Soliton Propagation in the Presence of 3rd-Order Dispersion," Opt. Commun. 114, 321-328 (1995); [CrossRef]
17. K. M. Hilligsøe, H. N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, "Initial steps of supercontinuum generation in photonic crystal fibers," J. Opt. Soc. Am. B 20, 1887 (2003); [CrossRef]
18. J. A. Gardecki and M. Maroncelli, "Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy," Appl. Spectrosc. 52, 1179-1189 (1998); [CrossRef]
19. A. Ortigosa-Blanch, J. C. Knight, and P. S. J. Russell, "Pulse breaking and supercontinuum generation with 200-fs pump pulses in photonic crystal fibers," J. Opt. Soc. Am. B 19, 2567-2572 (2002); [CrossRef]
20. T. M. Fortier, J. Ye, S. T. Cundiff, and R. S. Windeler, "Nonlinear phase noise generated in air-silica microstructure fiber and its effect on carrier-envelope phase," Opt. Lett. 27, 445-447 (2002); [CrossRef]
21. A. Apolonski, B. Povazay, A. Unterhuber, W. Drexler, W. J. Wadsworth, J. C. Knight, and P. S. Russell, "Spectral shaping of supercontinuum in a cobweb photonic-crystal fiber with sub-20-fs pulses," J. Opt. Soc. Am. B 19, 2165-2170 (2002); [CrossRef]
22. G. Genty, M. Lehtonen, H. Ludvigsen, J. Broeng, and M. Kaivola, "Spectral broadening of femtosecond pulses into continuum radiation in microstructured fibers," Opt. Express 10, 1083-1098 (2002); [PubMed]
23. . J. Bewersdorf and S. W. Hell, "Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz," J. Microsc. 191, 28-38 (1998); [CrossRef]

Appl. Spectrosc.

• J. A. Gardecki and M. Maroncelli, "Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy," Appl. Spectrosc. 52, 1179-1189 (1998); [CrossRef]

J. Microsc

• D. W. Piston, B. R. Masters, and W. W. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Microsc. 178 ( Pt 1), 20-27 (1995); [CrossRef]

J. Microsc.

• . J. Bewersdorf and S. W. Hell, "Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz," J. Microsc. 191, 28-38 (1998); [CrossRef]

J. Opt. Soc. Am B

• W. J. Wadsworth, A. Ortigosa-Blanch, J. C. Knight, T. A. Birks, T. P. M. Man, and P. S. Russell, "Supercontinuum generation in photonic crystal fibers and optical fiber tapers: a novel light source," J. Opt. Soc. Am. B 19, 2148-2155 (2002); [CrossRef]

J. Opt. Soc. Am. B

• J. M. Dudley, L. Provino, N. Grossard, H. Maillotte, R. S. Windeler, B. J. Eggleton, and S. Coen, "Supercontinuum generation in air-silica microstructured fibers with nanosecond and femtosecond pulse pumping," J. Opt. Soc. Am. B 19, 765-771 (2002); [CrossRef]
• K. M. Hilligsøe, H. N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, "Initial steps of supercontinuum generation in photonic crystal fibers," J. Opt. Soc. Am. B 20, 1887 (2003); [CrossRef]
• A. Ortigosa-Blanch, J. C. Knight, and P. S. J. Russell, "Pulse breaking and supercontinuum generation with 200-fs pump pulses in photonic crystal fibers," J. Opt. Soc. Am. B 19, 2567-2572 (2002); [CrossRef]
• A. Apolonski, B. Povazay, A. Unterhuber, W. Drexler, W. J. Wadsworth, J. C. Knight, and P. S. Russell, "Spectral shaping of supercontinuum in a cobweb photonic-crystal fiber with sub-20-fs pulses," J. Opt. Soc. Am. B 19, 2165-2170 (2002); [CrossRef]

J. Opt. Soc. Am. B.

• A. V. Husakou and J. Herrmann, "Supercontinuum generation, four-wave mixing, and fission of higher-order solitons in photonic-crystal fibers," J. Opt. Soc. Am. B 19, 2171-2182 (2002); [CrossRef]

J. Phys. D

• C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo -Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, "An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy," J. Phys. D 37, 3296 (2004); [CrossRef]

Jpn. J. Appl. Phys.

• K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, "Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source," Jpn. J. Appl. Phys. Part 2 44, L167-L169 (2005); [CrossRef]

Opt. Commun.

• J. N. Elgin, T. Brabec, and S. M. J. Kelly, "A Perturbative Theory of Soliton Propagation in the Presence of 3rd-Order Dispersion," Opt. Commun. 114, 321-328 (1995); [CrossRef]

Opt. Express

• G. McConnell, "Confocal laser scanning fluorescence microscopy with a visible continuum source," Opt. Express 12, 2844-2850 (2004); [CrossRef] [PubMed]
• G. Genty, M. Lehtonen, H. Ludvigsen, J. Broeng, and M. Kaivola, "Spectral broadening of femtosecond pulses into continuum radiation in microstructured fibers," Opt. Express 10, 1083-1098 (2002); [PubMed]

Opt. Lett.

• T. M. Fortier, J. Ye, S. T. Cundiff, and R. S. Windeler, "Nonlinear phase noise generated in air-silica microstructure fiber and its effect on carrier-envelope phase," Opt. Lett. 27, 445-447 (2002); [CrossRef]
• J. K. Ranka, R. S. Windeler, and A. J. Stentz, "Optical properties of high-delta air-silica microstructure optical fibers," Opt. Lett. 25, 796-798 (2000); [CrossRef]
• H. N. Paulsen, K. M. Hilligsoe, J. Thogersen, S. R. Keiding, and J. J. Larsen, "Coherent anti-Stokes Raman scattering microscopy with a photonic crystal fiber based light source," Opt. Lett. 28, 1123-1125 (2003); [CrossRef] [PubMed]

Phys. Med. Biol.

• G. McConnell and E. Riis, "Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2," Phys. Med. Biol. 49, 4757-4763 (2004); [CrossRef] [PubMed]

Phys. Rev. Lett.

• J. Herrmann, U. Griebner, N. Zhavoronkov, A. Husakou, D. Nickel, J. C. Knight, W. J. Wadsworth, P. S. Russell, and G. Korn, "Experimental evidence for supercontinuum generation by fission of higher-order solitons in photonic fibers," Phys. Rev. Lett. 88, 173901 (2002); [CrossRef] [PubMed]

Proc. Natl. Acad. Sci. USA

• W. R. Zipfel, R. M. Williams, R. Christie, A. Yu Nikitin, B. T. Hyman, and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci. USA 100, 7075-7080 (2003); [CrossRef] [PubMed]
• A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA 99, 11014-11019 (2002); [CrossRef] [PubMed]

Other

• "Photonic Crystal Fiber Data Sheet (NL-1.5-670)" (Crystal Fibre A/S), retrieved April, 2005, <a href="http://www.crystal-fibre.com/datasheets/NL-15-670.pdf">http://www.crystal-fibre.com/datasheets/NL-15-670.pdf</a>
• G. P. Agrawal, Nonlinear fiber optics, 3rd ed. (Academic Press, San Diego, 2001).

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