1. Introduction

Supercontinuum (SC) generation in highly nonlinear photonic crystal fibers (PCFs) with pulses from femtosecond lasers has led to the birth of a novel light source for microscopy. A PCF consists of a fused silica or air (hollow) core surrounded by cylindrical holes running along the length of the fiber. The optical properties of PCFs depend on the layout of the holes, the core size and shape [1]. Although the theory behind SC generation in PCFs is now well understood [2–6], its application to microscopy is still in its early stages.

To date, SC generation in PCFs has been used in coherent anti-Stokes Raman scattering (CARS) microscopy [7], confocal laser scanning fluorescence microscopy [8], confocal fluorescence lifetime imaging [9] and recently, two-photon excitation fluorescence (TPEF) microscopy [10, 11]. In their work, McConnell and Riis have performed two-photon microscopy on cells by employing frequency-doubled soliton radiation generated by a PCF [10]. Isobe and co-workers have demonstrated multi-excitation wavelength TPEF microscopy using the multi-peak NIR output of a PCF (700 nm to 950 nm) [11]. To the best of our knowledge, there has been no report on two-photon fluorescence microscopy using the visible nonsolitonic radiation of a PCF as a light source. This part of the PCF output spectrum is potentially interesting for TPEF; it affords direct short wavelength TPEF without the use of a doubling crystal.

Two-photon excitation fluorescence microscopy has been an invaluable tool for deep and live tissue intrinsic fluorescence imaging [12–14]. Several endogenous fluorophores are, however, excited with wavelengths between 250 nm and 320 nm including elastin, NADH and aromatic amino acids like tryptophan, tyrosine and phenylalanine. Unfortunately, efficient two-photon fluorescence imaging of cells and tissues using these fluorophores cannot be performed due to lack of suitable light sources that emit the necessary short wavelengths for two-photon excitation. A pulsed laser with fs or ps pulse width and an output wavelength in the visible region between 500 nm and 600 nm would be required. The tuning range of a typical femtosecond Ti:Sapphire laser is, however, limited to 700 nm to 1000 nm. Even when combined with a frequency doubling crystal, an additional tuning range between 350 nm and 500 nm is still unsuitable. To address this concern, we have developed a simple and robust method of generating tunable pulsed radiation at wavelengths between 500 nm to 600 nm based on the visible nonsolitonic radiation (NSR) from a highly nonlinear PCF excited with a femtosecond Ti:Sapphire laser. We believe that this is the first demonstration of two-photon fluorescence microscopy using the visible NSR from a PCF. We also show tunability of the NSR by varying the excitation intensity and wavelength. Furthermore, we show results on two-photon fluorescence spectroscopy and microscopy of tryptophan powder.

 

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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2. Experiments

A 13-cm long nonlinear PCF (NL-1.5-670, Crystal Fibre A/S) with a small silica core was used in the experiments (see fig 1) [15]. It has a core diameter of 1.5 μm, a NA of 0.24 at 780 nm, a zero-dispersion wavelength (ZDW) at 670 nm and a high nonlinear coefficient of 214 W^-1 km^-1 at 670 nm. From theoretical and experimental studies on PCFs, the efficiency of generating blue-shifted NSR depends mainly on the ZDW and the non-linearity of the fiber. The blue-shifted NSR is a result of the soliton decay, and its wavelength is determined by phase matching [16]. In an experimental study on a PCF with slightly different ZDW (660 nm) and core diameter (1.7 μm), NSR was efficiently generated in the 500 nm to 600 nm spectral region with excitation wavelengths between 700 nm and 800 nm. Their results were in agreement with calculations on the basis of phase matching [17].

To efficiently couple the excitation light (100 fs, 82Mhz, 700–800 nm) into the core of the PCF, an NIR AR-coated aspheric lens (f=3.1 mm, NA=0.68) was used. In order to investigate the spectral properties of the PCF, its output was coupled into a spectrometer (PC2000, Ocean Optics).

Figure 2 (left) shows the dependence of the SC spectrum on excitation power and wavelength. When, 720 nm NIR laser radiation was used to excite the PCF; spectral broadening was observed starting at a laser power of 3 mW. The observed broadening was centered at the excitation wavelength and increased for higher excitation powers.

At about 150 mW of excitation power, visible radiation centered near 550 nm was observed. This visible radiation is roughly 200 nm below that of the broadened NIR radiation. As the excitation power was increased, the visible radiation shifted towards the blue and its spectral width broadened. At much higher excitation intensities, the spectral gap between the broadened NIR radiation and visible radiation was filled up by low-intensity radiation.

Blue-shifting of the visible radiation peak was also observed by tuning the laser to longer excitation wavelengths (see Fig. 2 right). Adjusting the excitation wavelength from 700 nm to 740 nm caused the peak of the visible radiation to shift from 530 nm to 425 nm. This demonstrates that the visible radiation can be optimized for the excitation of different fluorophores.

In order to test the PCFs for short wavelength TPEF, we coupled the output of the PCF into a homebuilt two-photon microscope using an infinity corrected objective lens (Plan Fluor 40X/0.75NA, Nikon) and a short-wave pass filter to attenuate the wavelengths above 600 nm (see fig. 3). The typical power of the visible radiation (500 nm to 600 nm) measured after the short-wave pass filter was 5 mW. At this point, the visible radiation from the PCF was used as an excitation source for TPE. A short-wave pass dichroic beamsplitter (R>99% at 532 nm, T>75% at 325 nm -475 nm) was used to reflect the excitation light on to the sample and transmit the emission of the sample to the imaging spectrograph system. A microscope objective (Fluor 40X/1.30NA oil immersion, 160 mm tube length, Nikon, Japan) was used to focus the excitation light on to the tryptophan powder sample. The imaging spectrograph system consists of two prisms to disperse the emission, a UV-VIS achromat to focus the spectral components on to a CCD camera (Princeton Instruments, Spec-10:2KBUV), and a PC to acquire the spectra and analyze the spectral image. The spectral image acquisition time was limited by the CCD camera and amounted to about 2 minutes/frame.

 

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. 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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We experimentally measured the two-photon fluorescence spectrum of the tryptophan powder (Fig. 4 left). The data shown were corrected using the spectral transmission characteristics of the beamsplitter and the emission filter set. The measured tryptophan fluorescence spectrum, although spectrally narrower in width, was considered to be in fair agreement with the standard (one-photon) fluorescence emission of tryptophan with a peak at around 350 nm [18]. We attribute the difference to a poor correction for the transmission of the setup. Below 380 nm the efficiency of the setup, in particular of the objective, is rapidly going down. To confirm that the emission is a result of two-photon excitation, we measured the tryptophan emission intensity for varying excitation power. The slope of the linear fit of the double-logarithmic plot was found to be 2.16 implying a two-photon excitation process of the tryptophan.

 

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. 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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XY and XZ scan images of the tryptophan powder were recorded using the oil immersion objective (see Fig. 5). The XZ image clearly demonstrates the optical sectioning due to two-photon excitation. The images (224×224 pixels) were acquired at 2.1 ms per pixel (2 minutes/image). Despite the poor transmission of the microscope objective the average signal from the tryptophan grains was high. It amounted to approximately 10000 detected photons per pixel (integrated from 330–400 nm).

Some distortions in the images were observed, seen as dark lines, which we believe to be due to small fluctuations in the PCF output. We measured the stability of the PCF by recording a 100 s time trace of the (attenuated) output of the PCF using the spectrograph. The trace was recorded at 2 ms per data point and spectra were integrated from 330–400 nm. The relative intensity fluctuations and the power spectrum (Fourier transform of the relative intensity fluctuation) are shown in Fig. 6. The time trace shows rms fluctuation of about 3%.

3. Discussion and conclusion

Our experimental results on the spectral characteristics of the PCF with varying excitation power and wavelength are consis tent with the recent theoretical and experimental studies. When the excitation wavelength falls within the anomalous dispersion region of the PCF, i.e., above the (lower) ZDW, the initial steps of SC generation can be described as follows [17]: (1) the pulse is initially slightly broadened by self-phase modulation (SPM) and Raman scattering; (2) a red-shifted soliton is formed due to third-order dispersion and self frequency shifting; (3) it evolves toward a high-order soliton and because it is not stable, it breaks up into constituent first-order solitons [1, 19], generating blue-shifted nonsolitonic radiation at wavelengths determined by phase matching [16]; (5) finally, subsequent four-wave mixing (FWM) and Raman scattering enhance the formation of a broad SC. The blue-shift of the NSR in step 4 is a direct consequence of the red-shift of the evolving excitation pulse that leads to phase matching at a more blue-shifted wavelength. Consequently, an excitation pulse at a higher wavelength results in a lower wavelength NSR.

 

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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We have shown that the visible NSR from a PCF is a suitable light source for short wavelength TPEF microscopy. There are, however, two issues that need further attention. First, the stability of the PCF output is reasonable but not optimum for TPE. Because of the high nonlinearity of PCFs, the generated SC is very sensitive to the incident excitation amplitude fluctuations producing a complex phase distribution in the SC and additional noise [20]. One study found that reducing the fiber length results in less fluctuations and a smoother SC [21]. Secondly, the pulse duration of the visible NSR has to be determined. The difficulty in measuring the NSR pulse duration by conventional SHG-based autocorrelation technique lies on the low detection sensitivity between 250 nm to 300 nm. This may be due to low transmission of most optical elements and low detection efficiency of conventional detectors in this UV spectral region. Presently, we are implementing a TPE-based autocorrelator to measure the pulse duration of the visible NSR. We expect the NSR to have significantly broadened pulse duration with respect to the 100 fs pump pulses due to the highly dispersive nature and fast spreading in time of the NSR components. Previous simulations predicted the nonsolitonic radiation (superimposed waves between 500 nm to 600 nm) to be in the order of tens of picoseconds for 1 meter and a few picoseconds for a 10 cm PCF [22]. A picosecond pulse duration is, however, very well usable in TPEF microscopy [23].

Finally, our results demonstrate the potential of a PCF as a simple and inexpensive light source of visible pulsed light for two-photon fluorescence spectroscopy and microscopy. Further work is required to evaluate the potential of a PCF as a light source in TPEF microscopy of biological specimens.