Fiber Four-Wave Mixing Source for Coherent Anti-Stokes Raman Scattering Microscopy
Simon Lefrancois1,∗ , Dan Fu2 , Gary R. Holtom2 , Lingjie Kong1 , William J. Wadsworth3 , Patrick Schneider4 , Robert Herda4 , Armin Zach4 , X. Sunney Xie2 and Frank W. Wise1
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Department of Applied Physics, Cornell University, Ithaca, NY 14853, USA Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA 3 Centre for Photonics and Photonic Materials, University of Bath, Bath, BA2 7AY, UK 4 TOPTICA Photonics AG, 19 Lochhamer Schlag, Graefelﬁng (Munich) 82166, Germany ∗ Corresponding author: sl694@cornell.edu
Compiled March 3, 2013

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We present a ﬁber-format picosecond light source for coherent anti-Stokes Raman scattering microscopy. Pulses from an Yb-doped ﬁber ampliﬁer are frequency-converted by four-wave mixing in normal dispersion photonic crystal ﬁber to produce a synchronized two-color picosecond pulse train. We show that seeding the four-wave mixing process overcomes the deleterious eﬀects of group-velocity mismatch and allows eﬃcient conversion into narrow frequency bands. The source generates more than 160 mW of nearly-transform-limited pulses tunable from 775 to 815 nm. High-quality coherent Raman images of animal tissues and cells acquired with this source are presented. c 2013 Optical Society of America OCIS codes: 180.5655, 190.4970, 320.7140

Coherent anti-Stokes Raman scattering (CARS) microscopy allows label-free biological imaging by exciting intrinsic molecular vibrations [1]. However, it requires two synchronized picosecond pulse trains with bandwidths smaller than the relevant Raman linewidth and a frequency spacing tunable to the Raman shift of interest. This is generally accomplished using a solid-state Nddoped laser that is frequency-doubled to synchronously pump an optical parametric oscillator (OPO) [2]. Such bulk laser cavities are complex and require careful alignment and maintenance, limiting the use of CARS microscopy outside specialized laboratories. A turn-key source based on optical ﬁber technology would make CARS more accessible to its intended users. Two-color femtosecond ﬁber lasers can be built using soliton self-frequency shift [3]. To maximize spectral resolution and contrast, picosecond sources are desirable. A frequency-doubled ﬁber source can pump a bulk OPO [4]. A two-color Er-doped ﬁber system was realized using highly nonlinear ﬁber and periodically-poled lithium niobate (PPLN) [5]. Electronically synchronized actively modelocked ﬁber lasers provide rapid spectral tuning [6]. However, limited pulse energy in the former and longer pulse duration in the latter yield peak powers lower than from conventional solid-state systems. A major challenge is to ﬁnd a ﬁber-based frequencyconversion scheme scalable to high powers. Four-wave mixing (FWM) in photonic crystal ﬁber (PCF) has been used to convert 100-200 picosecond pulses to large frequency shifts [7, 8]. However, unseeded FWM leads to large deviations from the transform limit and signiﬁcant ﬂuctuations in the converted pulses [9], both of which are detrimental to CARS imaging. Transformlimited pulses with spectra that just ﬁll the vibrational linewidth (∼10 cm−1 ) would be optimal. For the desired

few-picosecond pulses, interaction lengths are only tens of centimeters due to group-velocity mismatch (GVM), which limits FWM conversion. As a result of these issues, CARS microscopy of biological samples has not been demonstrated with a ﬁber-FWM source. Here we present a ﬁber-based picosecond source for CARS microscopy. Frequency conversion is achieved by four-wave mixing in normal-dispersion PCF. Seeding the process mitigates GVM and suppresses noise. Pulses from a 1 µm ﬁber ampliﬁer are converted to around 800 nm with up to 160 mW of average power and durations around 2 ps. Frequency shifts in the range 2650 cm−1 to 3200 cm−1 have been achieved. We use this system to image mouse brain and skin tissues, as well as single cells. This is the ﬁrst ﬁber instrument to oﬀer performance comparable to solid-state systems. At normal dispersion, phase-matching of the FWM process yields widely-spaced and narrow bands, as required for CARS. Their position can be controlled by tailoring the dispersion of a PCF, mainly its zero-dispersion wavelength (ZDW). Fig. 1 shows the phase-matching diagram for an endlessly singlemode PCF with a ZDW of 1051 nm, calculated as in [10]. The dispersion coeﬃcients βn at 1036 nm are: 1.48 fs2 /mm, 59.5 fs3 /mm, -69.5 fs4 /mm, 136 fs5 /mm and -180 fs6 /mm. The continuous-wave (CW) pump power matches the expected pulse peak power. This shows that a pump laser tunable from 1030 nm to 1040 nm can be shifted by FWM in PCF to wavelengths between 770 nm and 820 nm with narrow bandwidths. To understand the FWM process in the pulsed regime, we perform numerical simulations [11]. The simulations account for higher-order dispersion, spontaneous and stimulated Raman scattering, self-steepening and input shot noise. With only the input picosecond pump and un-

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Fig. 1. (Color online) Phase-matched four-wave mixing gain for an endlessly singlemode PCF. The ZDW is 1051 nm, the non-linear parameter γ=9.6 (W·km)−1 and the CW power is P0 =3.6 kW.

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seeded sidebands, the process initially grows from spontaneous noise. Fig. 2(a) shows the resulting spectrum after the signal ﬁeld near 800 nm reaches 3.1 nJ of pulse energy, typically required for a CARS source. Broad (>10 nm), randomly-ﬂuctuating signal and idler bands develop. The signal energy saturates below 6 nJ as supercontinuum generation takes over due to non-phasematched processes dominating beyond the GVM length. The GVM places a clear limit on the FWM process. We propose that seeding the FWM process allows the ﬁelds to build up before GVM separates them. Seeding is known to reduce ﬂuctuations, but to our knowledge there is no prior report of its use to counter GVM. We simply inject CW light at the idler frequency. The resulting spectrum after 30 cm of propagation is compared with the unseeded case at similar energies in Fig. 2(a) and details are shown in Figs. 2(b)-(c). Signiﬁcant narrowing is achieved and the conversion eﬃciency is above 10%. Further conversion is limited by coherent energy exchange between ﬁelds, which generates structured pulses. The experimental setup is shown in Fig. 3. A tunable Yb-doped ﬁber laser (modiﬁed TOPTICA PicoFYb) is coupled to a divided-pulse ampliﬁer based on 10 µm core diameter double-clad Yb-doped ﬁber [12]. This produces 2.5 W of pulses with 7.7 ps duration at 54 MHz repetition rate. This is combined with a ﬁber-coupled diode laser tunable from 1400 nm to 1490 nm and providing up to 30 mW (TOPTICA DLpro). The polarization-matched beams are coupled into an endlessly-single-mode PCF we fabricated and matching the ﬁber above. Filters block the anti-Stokes light generated by mixing of the signal and pump in the PCF [13]. A fraction of the 1 µm pulses is picked-oﬀ before the PCF and combined with the polarization-matched signal at the microscope. Experimental results with 30 cm of PCF are shown in Fig. 4. 1.6 W of pump pulses are coupled into the PCF. The idler is seeded with 5.1 mW at 1471 nm. The generated signal pulses have 166 mW of average power and a duration of 1.8 ps, while 308 mW of pump pulses are picked-oﬀ. The performance is similar to solid-state systems [2]. Higher powers or longer ﬁbers yield higher 2

Fig. 2. (Color online) Simulated FWM. (a) Full spectrum without idler seeding after propagation through 56 cm (light solid) and 2 m (light dotted), and with idler seeded after 30 cm (black solid). GVM length is ∼50 cm. (b) Seeded FWM: signal (solid), pump (dotted) and idler (dashed) pulses. (c) Signal spectrum. The input pulse is centered at 1036 nm with 7.5 ps duration and 3.6 kW peak power. Idler seed power is 5 mW at 1470 nm.
HWP PBS Fiber laser Tunable CW laser LP HWP Out PCF SF SP Delay HWP

Fig. 3. (Color online) Experimental setup. HWP: halfwave plate, PBS: polarizing beamsplitter, LP: longpass dichroic mirror, SF: ﬁlters (Thorlabs DMSP1000, Chroma HQ735LP), SP: short-pass dichroic mirror.

signal energies and structured spectra. The frequency diﬀerence of 2850 cm−1 corresponds to the CH2 stretch. With the tuning range of the diode, we achieved similar performance for frequency shifts of 2650 cm−1 and 2950 cm−1 . By use of a similar ﬁber ampliﬁer centered at 1031 nm and an ampliﬁed diode laser tuned to 1546 nm, we generated a signal wavelength of 774 nm, corresponding to a Raman shift of 3200 cm−1 . Coarse tuning can be accomplished by changing the pump wavelength, while ﬁne tuning over 1 to 2 nanometers can be done by tuning the seed. No re-alignment is required. The FWM signal and picked-oﬀ pump are coupled into a laser-scanning microscope (customized Zeiss LSM 510) and focused using a 40X water-immersion objective with a numerical aperture of 1.1. We detect the forwardgenerated CARS signal with a non-descanned photomultiplier tube. The total power delivered to the samples is

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Fig. 4. (Color online) Experimental FWM results after 30 cm of PCF: signal (a) spectrum and (b) autocorrelation, (c) idler and seed. (d) Picked-oﬀ pump spectrum. about 60 mW. CARS images at a 2850 cm−1 shift from a mouse ear are presented in Figs. 5(a)-(b). The former shows the stratum corneum at the skin surface, and the latter reveals the sub-cellular lipid distribution in a sebaceous gland 40 µm deep in tissue. Fig. 5(c) shows a mouse brain section with myelin sheath wrapped around axons. Finally, Fig. 5(d) shows isolated rat ﬁbroblast cells. The typical lipid droplet signal relative to noise is 40 for cell images and 80-120 for sebaceous glands, conﬁrming image quality. At a shift of 2950 cm−1 , this dropped to 25 for cells and 40 for sebaceous glands.
(a) (b)

ring (SRS) imaging [14]. Preliminary experiments with dodecane showed excessive ﬂuctuations in the SRS signal, precluding imaging. No attempt was made to design the present source for low noise, and parametric processes such as FWM amplify the noise of the pump and the seed. We can conclude that ﬁber sources will have to be designed for low-noise performance for SRS microscopy. Optimization of the detection scheme will also be desirable, for instance by modulating the FWM signal and detecting the quieter ﬁber laser pulse train. To summarize, we have demonstrated a ﬁber-based source for CARS microscopy based on picosecond FWM in PCF. Seeding of the FWM process overcomes the limitations of noise and group-velocity mismatch. More than 160 mW of 2 ps pulses can be generated at suitable frequency shifts covering more than 500 cm−1 . The system could be further integrated using specialty splices and ﬁber couplers. This is a signiﬁcant step toward a turnkey ﬁber-based source that will allow CARS microscopy to extend into biological and clinical applications. This work was supported by the National Institute of Health (EB002019) and the National Science Foundation (BIS-0967949). We thank B. G. Saar and C. W. Freudiger for help with microscopy and insightful discussions. References
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Fig. 5. (Color online) Forward-CARS images at 2850 cm−1 : (a) stratum corneum and (b) sebaceous gland in mouse ear, (c) mouse brain section, (d) rat ﬁbroblasts. 512x512 pixels at 4 s/frame, no averaging. We attempted to perform stimulated Raman scatte3

Informational Fourth Page References
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