Abstract

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

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References

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Appl. Spectrosc.

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. 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

Opt. Lett.

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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Figures (6)

Fig. 1.
Fig. 1.

SEM image of the PCF (NL-1.5-670, Crystal Fibre A/S) “holey” region (left) and the core (right) [15].

Fig. 2.
Fig. 2.

Left: Output spectrum of the PCF (length=13 cm) with increasing laser input power (excitation wavelength=720 nm). The white dashed line denotes the zero-dispersion wavelength (ZDW) of the PCF. Right: Output spectrum of the PCF with varying laser input wavelength (excitation average power=300 mW).

Fig. 3.
Fig. 3.

Two-photon excitation fluorescence microscope setup coupled with the PCF. The inset shows the visible non-solitonic radiation generated along the 13-cm long PCF when excited with near infrared laser.

Fig. 4.
Fig. 4.

Left: Experimentally measured (circles) and standard (solid line) fluorescence spectra of tryptophan powder [18]. Right: Double-logarithmic plot of the tryptophan powder fluorescence intensity versus excitation intensity. The slope of the best -fit line is 2.16.

Fig. 5.
Fig. 5.

XY scan (left) and XZ scan (right) two-photon excitation fluorescence images of tryptophan powder using a Fluor 40X/1.30NA oil immersion objective lens.

Fig. 6.
Fig. 6.

Upper: Time trace of the relative intensity fluctuations of the PCF output. Lower: Power spectrum obtained by Fourier transforming the time trace (50,000 points).

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