We demonstrate a homodyne coherent anti-Stokes Raman scattering (CARS) technique based on femtosecond laser pulse shaping. This technique utilizes fast phase cycling to extract nonlinear Raman signatures with a self-generated reference signal acting as a local oscillator. The local oscillator is generated at the focus and is intrinsically stable relative to the Raman signal even in highly scattering samples. We can therefore retrieve phase information from the Raman signal and can suppress the ubiquitous non-resonant background.
©2010 Optical Society of America
The vibrational level structure of biomolecules could provide highly specific structural, metabolic and functional contrast in tissue, but infrared spectroscopy in tissue is generally hindered by strong water absorption. Raman scattering using visible or near-IR light can occur much deeper in tissue, but Raman cross sections of intrinsic markers are generally small and sensitive measurement techniques are required for biologically relevant concentrations . Spontaneous and coherent Raman effects imprint the vibrational spectrum onto light scattered from the sample, and both techniques can be used to obtain spectroscopic information from tissue , but coherent anti-Stokes Raman scattering (CARS) imaging offers several advantages: the nonlinearity provides inherent optical sectioning, and the generated higher-energy anti-Stokes signal is not contaminated by background fluorescence . However, non-resonant four-wave mixing (FWM) processes generally produce a strong, non-specific background at the anti-Stokes frequency, which results in distorted line shapes and a loss of imaging contrast. Techniques such as polarization-sensitive CARS or time-delay CARS (for a review see Ref .) can reduce or suppress this ubiquitous background, but these techniques suppress the non-resonant components at the expense of a reduction of the generally already much smaller resonant components.
Here we demonstrate a new approach to cleanly and efficiently detect CARS signals. In common with other recent work [4–12] we distinguish resonant from non-resonant polarization by taking advantage of the difference in their phase properties: the non-resonant susceptibility is purely real, while the resonant susceptibility is complex. A reference anti-Stokes field of a determined phase (the local oscillator (LO)) can extract the imaginary part of the resonant contribution background-free by homodyning the signal , and the local phase at the focus can be measured periodically to compensate for variations caused by scattering . Pulse-shaping approaches of varying complexity have also been implemented [6–12]; for example, one approach uses the real, non-resonant polarization generated at the focus within the sample as a local oscillator to interfere with the resonant component that is appropriately phase-shifted by a pulse shaper , then uses a high resolution spectrometer to extract the background-free Raman contribution. The drawbacks of the existing techniques are that they either rely on a local oscillator that is supplied externally to the sample [4,7–9], or uses a monochromator to spectrally analyze the Raman contributions [6,10,11]. Because of the wavelength difference between the CARS generating beams and the LO, an externally supplied LO experiences a different effective propagation path (and fluctuations thereof), leading to an unstable interference with the generated CARS radiation. The use of a monochromator is troublesome because its performance is inevitably degraded by scattering. In contrast, our approach uses a femtosecond pulse shaping technique with rapid update rates [13,14] to generate both a static non-resonant LO and a phase-rotating resonant contribution at the focus within the sample. These two contributions result in an inherently stable interference that can be recorded with a detector and a lock-in amplifier without the need for a high-resolution spectrometer.
The principle of the phase-cycling CARS is illustrated in Fig. 1(a) . The broad bandwidth pulse that serves as the degenerate pump/probe pulse is shaped such that the phase ϕ of a narrow portion in the center of the spectrum is incremented from one pulse to the next (the rotating portion). The spectral width of the rotating portion and the bandwidth of the Stokes pulse are much narrower than the pump bandwidth and are on the order of the Raman linewidth. The two pulses interact with the susceptibility , where the non-resonant susceptibility is real and assumed to be frequency independent and the Raman susceptibility is complex and depends on the difference Ω = ωϕ − ω s between the pump and the Stokes frequencies. Although here we place the rotating portion in the center of the pump spectrum (ωϕ = ω p), its exact position is not critical.
The interaction within the sample leads to static and phase-rotating polarizations. The static polarization is dominated by the non-resonant component (four-wave mixing (FWM)), which scales as , where Ap and As are the square root of the intensity of the pump/probe and Stokes pulses, respectively. This non-resonant component gives rise to a broadband four-wave mixing signal centered at the anti-Stokes frequency 2ω p-ω s, which constitutes the static LO. The phase-rotating polarization has components originating from both χnr and χr. χnr generates two identical components (one for which the phase-rotating portion serves as the pump and the static portion as the probe and one with the reverse order, respectively), which scale as , where Aϕ is the square root of the intensity of the phase-rotating spectral component. The dominant resonant component originates from the molecular coherence created by the phase-rotating portion and the Stokes pulse, which is then probed by the broad static portion. This resonant component gives rise to a broadband anti-Stokes Raman signal centered at ω p + ωϕ - ω s, where ωϕ = ω p is the frequency of the narrow rotating portion. We measure both quadratures of the phase-rotating component
This phase-cycling CARS method is not a broadband multiplex technique, in the sense that it does not resolve multiple Raman transitions in a single shot; selectivity here is achieved by tuning the frequency difference between the narrow-band Stokes and the narrow-band rotating portion in the broad pump spectrum (ωϕ − ω s). When this frequency difference coincides with a Raman transition, we obtain an absorptive peak in the imaginary channel and a dispersive feature in the real channel.
3. Experimental setup
The experimental setup for our phase-cycling CARS technique is shown in Fig. 1(b). A regenerative amplifier (Coherent, RegA) operating at 20 kHz was used as the laser source (~60 fs pulses at 800 nm), where 80% of its output pumped an optical parametrical amplifier (Coherent, OPA 9450) to generate a broad-band pump/probe beam (~80 fs pulses at 740 nm). This beam was then spectrally shaped with a 4-f pulse shaper based on an acoustic-optical modulator arrangement . The phase of the central portion of this spectrum (~0.6 nm wide) was rotated at a rate of 5 kHz. The remaining portion of the RegA output was converted to the narrow-band Stokes beam (0.6 nm spectral width, ~900 fs pulse length) by another 4-f pulse shaper acting as a spectral filter. Both beams were combined with a dichroic mirror and focused into the sample cuvette with a microscope objective (NA 0.25) resulting in a focal size of about 1.1 μm. The anti-Stokes light was separated from the transmitted beam with a 700 nm short-pass filter, detected with a biased photodiode, time-gated with a boxcar integrator (Stanford Research Systems, SR250), and measured with a lock-in amplifier (Stanford Research Systems, SR830) with a time constant of 1 s. To acquire a CARS spectrum the frequency of the narrow-band Stokes pulse was tuned by sweeping the spectral filter through the broad spectrum of the 800 nm input laser.
To demonstrate the concept of phase-cycling CARS, we measured the CARS spectrum of various solutions of benzene in carbon disulfide (CS2). Benzene has a pronounced Raman transition at 992 cm−1 (ring-stretch mode), while CS2 has no strong modes in the region from 750 cm−1 to 1300 cm−1 (CS2 and benzene have weak Raman transition at 800 cm−1 and 1200 cm−1, respectively). For comparison, we also acquired standard CARS spectra with narrow-band (about 0.6 nm) pump/probe and Stokes pulses. In order to account for the variation in power when tuning the Stokes wavelength we normalized the acquired traces by the signal from pure CS2.
Figure 2(a) shows the narrow-band CARS spectrum for benzene (100% volume concentration) with a peak at around 1000 cm−1, which is distorted by the interference between real and imaginary susceptibilities. Decreasing the concentration leads to decreased signal-to-background ratios and more severe line-shape distortions. For volume concentrations less than 10%, the resonant signal is almost indistinguishable from the large non-resonant background. In comparison, Fig. 2(b) and 2(c) show the real and imaginary channel, respectively, obtained with phase-cycling CARS. As indicated by Eq. (1), the real channel signal is a combination of both resonant and non-resonant contributions, resulting in an offset dispersive line shape of the Raman transition. In contrast, the imaginary signal is exclusively due to the resonant contribution, and thus shows an absorptive peak-like feature similar to a spontaneous Raman spectrum.
According to Eq. (1), the signal of our measurement should scale linearly with the Stokes power and quadratically with the pump power. These scaling behaviors are confirmed with measurements based on 10% volume concentration solution as shown in Fig. 3(a) . Equation (1) also predicts that for small benzene concentration the phase-cycling CARS signal scales linearly with concentration, which we confirmed with measurements shown in Fig. 3(b). Here the uncertainty (error bars in Fig. 3(b), given by the fluctuation of the lock-in amplifier) is almost constant for different volume concentrations. It limits the sensitivity of current experimental setup to 0.5% volume concentration (when the signal is equal to the uncertainty), corresponding to about 108 benzene molecules in the focal volume. This uncertainty is largely dominated by our detection electronics (primarily by the boxcar amplifier), which prevents us from pursuing shot-noise limited detection. We expect a substantial improvement by adapting this phase-cycling CARS approach to a rapid pulse shaping technique for modelocked lasers recently developed in our group .
To further explore the properties of the phase-cycling CARS technique, we compared our experimental result with numerical calculations, as shown in Fig. 4(a) and 4(b). In these figures we notice weak, broad, negative wings in proximity to the narrow Raman line. While the narrow line results from the convolution of the narrow phase-rotating portion with the sharp Raman line, the broad wings originate from convolutions that contain at least one broad component: either the broad static portion with the narrow Raman line or the narrow phase rotating portion with the broad non-resonant background. In order to minimize these wings, we can refine the phase profile of the rotating portion of the pump/probe pulse by inserting a π phase step in the center of the rotating portion [16,17]. The phase step in the rotating portion results in a phase step in the broad signal contributions. As a result, these contributions cancel when integrated over the entire CARS spectrum; hence the broad wings are eliminated. Because the introduced phase step can enhance or counteract the inherent change in the phase profile of a Raman resonance depending on their relative phase, lock-in detection can selectively extract the narrow resonant Raman contribution (Pϕ,r). In contrast to , however, it is not required to closely match the details of the Raman phase profile (we approximate it with a π phase step), easing the demands on the pulse shaper. Figure 4(c) and 4(d) show calculations and experimental data for such a phase shape. Here the width of the phase-rotating component is about 20 cm−1 with the π phase step at its center. The reduction of the width of the negative wings in the imaginary channel and the suppression of the large non-resonant offset in the real channel are apparent. The small offset in the real channel of the experimental data in Fig. 3(d) are likely due to a slightly asymmetric shape of our pump/probe spectrum.
The femtosecond pulse shaper effectively creates a combination of a picosecond pulse (the rotating narrow portion) and a femtosecond pulse (the static pump), and in this sense the CARS application is similar to the ps-fs scheme reported in ref , where these two pulses are out of phase (both have fixed inter-pulse phase) such that the nonresonant background generated from them cancel out if their relative spectral amplitude is carefully matched. Meanwhile, due to its much narrower spectral distribution, the picosecond pulse generates a much larger resonant signal than the femtosecond pulse. As a result, the total resonant signal is not balanced and does not cancel, and background free measurement is achieved. In our case, however, our ability to dynamically rotate the phase of a narrow portion of a broadband pump lets us retrieve both dispersive (real channel) and absorptive (imaginary channel) Raman features. In addition, precise amplitude matching between the picosecond and the femtosecond pulse is not necessary. Finally, while the ps-fs scheme is a broadband CARS technique that relies on a spectrometer to obtain spectral resolution, the spectral resolution of our phase-cycling CARS is determined by the frequency difference between the narrow band Stokes and the narrow band rotating component of the pump without the need for spectral analysis of anti-Stokes radiation.
The local oscillator that is used in our technique for homodyning is spectrally much wider than the Raman lines to be investigated. This ensures that even in congested regions of the Raman spectrum the local oscillator does not vary substantially when probing different Raman frequencies. The presence of several Raman lines within the broad bandwidth of the pump pulse does not produce interference cross terms, but merely superimposes the individual lines, as demonstrated in Fig. 5 .
It is instructive to compare the relative signal strengths of our phase-cycled CARS with those obtained by conventional narrow-band CARS. For a large nonresonant susceptibility (as is present in low-concentration samples) narrow-band CARS as a function of detuning yields a dispersive line shape with a large nonresonant offset. We therefore regard the difference between the maximum and the offset as the usable signal amplitude. For spectrally narrow input pulses, the amplitude of this signals scales as . Here, δ is the spectral width of the pump and Stokes pulses and E p and E s are the pulse energy of the pump and Stokes pulses, respectively. For phase-cycled CARS the measurement is background-free and the peak amplitude is used for comparison. In this case, the absolute signal scales as . Here, δ denotes the spectral width of the Stokes pulse and the width of the phase rotating spectral portion. The width of the pump pulse was assumed much larger than δ. Figure 6 compares the two signal amplitudes as a function of δ on the same scale.
In this figure, both amplitudes were normalized by the pulse energies E p 2 E s. We can see that for spectral widths on the order of the Raman line width, both techniques yield similar signal strengths for equal pulse energies. While spectrally narrow excitation pulses more efficiently excite Raman transitions, the broad pulses amplify weaker Raman signatures with a strong local oscillator.
Our technique could offer several potential advantages for tissue imaging. The local oscillator used for amplification does not have to be supplied, but is generated from ubiquitous, non-resonant four-wave mixing processes at the focus (co-localized with the Raman signal), which provides a stable phase reference even in a highly scattering environment. For weak Raman scatterers, the out-of-phase component provides absorptive Raman signatures, while the in-phase component provides essentially the strength of the LO. In the case of non-uniform four-wave mixing generation (i.e. if FWM itself generates contrast) the in-phase component could be used for normalization of the Raman contrast. Although we cannot record multiple Raman transitions in a single shot, selecting different frequencies out of a broadband Stokes spectrum produces different vibrational contrast, and very rapid update is possible. Our current experimental setup uses an amplified laser system with a pulse-shape repetition rate of 20 kHz, with which high-speed imaging of biological tissue is not feasible. Also, the threshold of photodamage of tissue samples is easily reached due to high peak intensity of the laser, especially since the broad spectral contribution in the pump/probe beam (corresponding to a temporally short pulse) creates a higher peak intensity pulse than in the narrow-band CARS case. Therefore, if the damage threshold of the sample under study is determined by peak intensity, rather than average power, the signal will be reduced. However, we have recently extended the pulse shaping technique to 80 MHz modelocked lasers with intrinsically lower peak intensity , which (when combined with a suitable dual color source) should make this technique suitable for high-speed imaging applications in tissue.
We described a homodyne coherent anti-Stokes Raman scattering technique based on femtosecond laser pulse shaping (phase-cycling) and demonstrated it with an amplified laser and AOM based pulse shapers. This technique utilizes a self-generated non-resonant background as a local oscillator to retrieve phase information of the Raman signal. This technique should offer high immunity to scattering and could therefore be applicable to imaging vibrational contrast in highly scattering samples like tissue.
This work was supported by the National Institutes for Health (1RC1CA145105) and funding from Duke University.
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