Using a low-cost microchip laser and a long photonic crystal fiber taper, we report a supercontinuum source with a very efficient visible conversion, especially in the blue region (around 420 nm). About 30 % of the total average output power is located in the 350–600 nm band, which is of primary importance in a number of biophotonics applications such as flow cytometry or fluorescence imaging microscopy for instance. We successfully demonstrate the use of this visible-enhanced source for a three-color imaging of HeLa cells in wide-field microscopy.
© 2010 Optical Society of America
Sub-nanosecond microchip lasers at 1064 nm are great pump candidates for producing supercontinuum sources, because of their high peak power, low cost and compactness. The association of sub-nanosecond 1064 nm lasers with highly nonlinear photonic crystal fibers (PCFs) has proved to be a technology mature enough to allow their commercialization  and their implementation in bio-medical systems for instance [2–4]. In fluorescence imaging microscopy or flow cytometry applications, as in many other ones, the whole visible spectrum is of main interest, because many fluorochromes (such as nucleic acid probes, fluorescent proteins, organic molecules or reactive and conjugated probes) have absorption bands. However, this spectral region (below 450 nm) proved to be tricky to reach in supercontinuum generation experiments, because of its very large detuning from the pump wavelength. The main physical reason for this limitation is well identified: the short-wavelength edge of the supercontinuum is imposed by a group-velocity matching condition between solitons in the infrared and trapped dispersive waves in the visible [5–7]. Based on the understanding of this mechanism, several approaches have been employed to further downshift the short-wavelength supercontinuum edge. They include using two PCFs with sequentially decreasing ZDW , PCF tapering with few meterlong transitions , PCF post processing , dual-wavelength pumping [12, 13], or simply optimizing of the group index matching condition with help of high-Δ PCFs . In all these demonstrations however, the pump power was converted into an ultra-broad supercontinuum, making the spectral power density in the blue region relatively low.
In this paper, we report the generation of a white-light supercontinuum with a very efficient visible conversion in the the 350–600 nm band and a maximum spectral power density in the blue (around 420 nm). This was achieved using a simple microchip laser at 1064 nm and a 10 m-long high-Δ PCF taper. We then demonstrate the great suitability of our source for biological imaging by implementing it on a wide-field fluorescence microscope. Three fluorochromes were excited at 385, 410 and 530 nm, allowing imaging of three distinguishable components inside HeLa cells. This demonstration was possible thanks to the high power transfer from the pump to the ultraviolet/visible part of the supercontinuum spectrum.
2. Fiber fabrication and experimental setup
The PCF used in the experiments was designed to combine the benefits of decreasing ZDW along length [8, 10] and high-Δ cladding structure , which are both known to produce a blue/UV-enhanced supercontinuum [7,10]. Our PCF sample is made of a 3 m-long section with a uniform diameter of 160 µm followed by a 7 m-long section along which the outer diameter decreases from 160 µm to a final diameter of 67 µm. Figure 1(a) and (b) shows the scanning electron microscope (SEM) images of the input and output fiber ends, respectively, with the same scale. The drawing parameters were suitably adjusted so that the cladding structure was preserved along the taper section, i.e. the d/Λ ratio was constant (d and Λ being respectively the hole diameter and hole-to-hole distance). The outer diameter evolution recorded during the fiber drawing process was almost linear in the taper section, except for the last 2 m, as shown in Fig. 1(c). The attenuation of the 10 m-long PCF taper was measured to be 3 dB at 1064 nm. The dispersion properties at the input and output of the PCF taper were measured in short fiber pieces using a low-coherence interferometry setup. The group index measured as a function of wavelength in the uniform section (at the PCF input) and at the taper end (at the PCF output) are represented in Fig. 2(a), in blue and red circles, respectively. The solid curves correspond to the group index curves computed with a finite element method, which are in excellent agreement with measurements. They depict the typical asymmetric U-shape, with the short-wavelength branch being much steeper because of strong silica dispersion in this spectral region. As the fiber outer diameter decreases, the group index curve is not significantly modified at short-wavelengths (because material dispersion dominates), but the long-wavelength branch significantly shifts towards short wavelengths. The corresponding group-velocity dispersion (GVD) curves deduced from the group index measurements and simulations are represented in Fig. 2(b) by circles and solid lines, respectively, in blue and red color for the PCF input and output, respectively. The ZDW is located at 970 nm at the fiber input, and decreases to 800 nm at the fiber output. Note that for wavelengths shorter than 1750 nm, the total dispersion increases as light propagates along the taper, while it decreases for wavelengths higher than 1750 nm. This PCF taper was pumped with a linearly polarized Q-switch microchip laser at 1064 nm delivering 0.6 ns pulses (FWHM) at a repetition rate of 7 kHz. The light was launched into the PCF taper with a ×20 microscope objective and the pump peak power effectively launched into the fiber was 5.8 kW (corresponding to an average power of 43 mW). The output spectrum was recorded with an optical spectrum analyzer (OSA) and cross-checked with a visible spectrometer operating between 250 and 900 nm (Ocean Optics HR4000).
3. Results and discussion
The black line in Fig. 3(b) shows the spectrum measured in the 10 m long PCF taper with the OSA set to a 1 nm resolution. The fiber output delivers a bright white-light spot, as can be seen from the inset picture in Fig. 3(b). As expected from such tapered PCFs , the generated supercontinuum spans from 350 to more than 1750 nm with an average output power of 17 mW. However, the main difference between our results and previous ones concerns the much more efficient generation of visible light, especially in the 350–600 nm spectral range. The average power measured over this spectral range with a series of spectral filters is 5 mW. The spectral power density was calculated assuming that the supercontinuum extends up to 2200 nm, by integrating the whole spectrum and normalizing to the measured output average power. It is maximum around 420 nm where it reaches 35 µW/nm, which is much higher than in the infrared region, as can be seen from Fig. 3(b) (top curve). The mode properties in the visible were investigated by imaging the fiber end face on a camera. The near-field mode profiles recorded after 10 nm bandpass filters are displayed in Fig. 3(c) as a function of wavelength. These images show that the visible light generation occurs in the fundamental fiber mode, which is of primary importance for a large number of potential applications. In a second set of experiments, the 10 m long PCF taper was cut into two parts after the 3 m-long uniform section, and the output spectrum was recorded. Figure 3(a) shows the fiber profile (in color) as a function of length corresponding to each spectrum in Fig. 3(b). Light was first launched into the remaining 7 m-long tapered section, and the corresponding spectrum is displayed in blue line in Fig. 3(a). A supercontinuum as broad as in the previous case is obtained. Nevertheless, the spectral features are very different in both cases. Indeed, the pump conversion into the supercontinuum is less efficient, and the spectral power density in the 350–600 nm range is reduced by a factor of nearly two, as compared to the 10 m-long fiber case. This means that the initial 3 m-long section plays an important role in the visible light generation , which can be understood as follows. In the long-pulse pumping regime, the short-wavelength generation is well explained by the presence of trapped dispersive waves emitted from solitons [7, 14]. These fundamental solitons are generated through modulation instability (MI) and progressively red-shifts due to intrapulse Raman scattering. The role of the 3 m-long section is thus to allow high peak power solitons to form from MI. This is illustrated experimentally by the red curve in Fig. 3(b), which corresponds to the spectrum recorded in the 3 m-long uniform fiber alone. This curve shows that the spectral broadening mainly occurs towards the long-wavelength side, which is characteristic of soliton formation from MI and subsequent intrapulse Raman scattering. This Raman supercontinuum obtained within the first few meters of propagation is thus mainly made of high peak power solitons. Once these solitons enter the tapered section, the most red-shifted ones experience a decreasing dispersion (for wavelengths higher than 1750 nm in Fig. 2(b)) combined with an increasing nonlinear coefficient , which allows compression, and thus increases the solitons peak power. This leads to an enhancement of the soliton self-frequency shift process, which consequently increases the spectral power density for wavelengths higher than 1750 nm. The global energy shed away by solitons to blue-shifted trapped dispersive waves is thus enhanced, which eventually causes the formation of the strong visible peak centered at 420 nm (see Fig. 3(b), top curve). Simultaneously, this phenomenon is assisted by the group index matched wavelengths which progressively shifts toward the blue as can be seen from Fig. 2(a), and allows a further down-shift of trapped dispersive waves to the blue region .
4. Demonstration of three-colors fluorescence imaging
In order to demonstrate the potential of this source for biomedical imaging, it was implemented on a commercial wide-field microscope (Nikon Ti-E). In wide-field microscopy, the excitation source is focused in the back focal plane (BFP) of the microscope objective lens in such a way that the collimated beam illuminates a large sample area (around 5000 µm2). This allows to record single-shot images directly on a camera. Thanks to the broad visible spectrum of our source, it is possible to record images with many labeled biological components. We limit here our investigation to three biological components inside human cancer cervical cells (HeLa cells). The cells nuclei were labeled with DAPI which is excited in the ultraviolet and emits around 450 nm. Rab6 proteins were stained using Green Fluorescent Protein (GFP) whose excitation and emission peaks are located around 485 nm and 510 nm respectively. The cells cytoskeleton (F-actin filaments) were labeled with Rhodamin Phalloidin which has excitation peak around 550 nm and emission peak around 580 nm. The white-light from the fiber output was collimated with an objective lens (×10) and focused onto the BFP of the microscope objective lens (×63, NA 1.49). Each colored components was imaged separately using appropriate commercial fluorescence filters sets from Semrock (DAPI-1160A, GFP-3035B and Cy3-4040B). The excitation wavelengths of the spectrally filtered supercontinuum source were centered at 385, 470 and 530 nm using these filter sets, and were typically 20 to 40 nm wide. Each biological components was observed with an EM-CCD camera. The cells nuclei, Rab6 proteins and F-actin pictures successively acquired are displayed in Fig. 4(a), 4(b) and 4(c) respectively, for a fixed gain and exposure times of 10, 500 and 4 ms. The fluorescence signal was collected over 50 nm spectral bands centered respectively around 450, 520 and 590 nm. Figure 4(d) shows the merge image reconstructued from the three separate images showed above. These images illustrate the great suitability of our supercontinuum source for fluorescence imaging, thanks to its enhanced spectral power density in the visible region, which corresponds to the absorption band of many useful fluorochromes used in biology.
We have experimentally demonstrated a supercontinuum source with a very efficient power transfer from the pump to the visible region between 350 and 600 nm, with a maximum spectral power density around 420 nm. This has been done using a low-cost sub-nanosecond microchip laser at 1064 nm pumping a 10 m-long PCF with a 7 m-long section tapered down from 160 to 67 µm outer diameter. The role of the initial uniform fiber section has been shown to be important in the optimization of the blue transfer. We have also demonstrated the great interest of this supercontinuum source in fluorescence imaging microscopy with visible fluorescent probes. This was done by implementing it on a wide-field fluorescence microscope and by successfully performing a three color imaging at 385, 470 and 530 nm, which showed three distinguishable components inside HeLa cells.
We acknowledge C. Spriet, A. Leray, L. Héliot from IRI (Université Lille 1) for fruitful discussions, R. Habert for technical assistance, and the group led by B. Goud (Institut Curie), and in particular S. Bardin, for providing the Rab6-GFP expressing HeLa cell line. This work was partly supported by the “Conseil Régional Nord Pas-de-Calais” and the “FEDER”.
References and links
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