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Short-wavelength two-photon excitation fluorescence microscopy of tryptophan with a photonic crystal fiber based light source

J. A. Palero, V. O. Boer, J. C. Vijverberg, H. C. Gerritsen, and H. J. C. M. Sterenborg

Open Access Open Access

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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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].

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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).

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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.

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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.

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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.

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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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