We present an experimental demonstration of single-pulse coherent anti-Stokes Raman spectroscopy (CARS) using a spectrally shaped broadband laser that is delivered by an optical fiber to a sample at its distal end. The optical fiber consists of a fiber Bragg grating component to serve as a narrowband notch filter and a combined large-mode-area fiber to transmit such shaped ultrashort laser pulses without spectral distortion in a long distance. Experimentally, our implementation showed a capability to measure CARS spectra of various samples with molecular vibrations in the fingerprint region. Furthermore, CARS imaging of poly(methyl methacrylate) bead samples was carried out successfully under epi-CARS geometry in which backward-scattered CARS signals were collected into a multimode optical fiber. A compatibility of single-pulse CARS scheme with fiber optics, verified in this study, implies a potential for future realization of compact all-fiber CARS spectroscopic imaging systems.
© 2017 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Coherent anti-Stokes Raman scattering (CARS) spectroscopy is a molecular analysis tool which has found numerous applications in combustion diagnostics, materials research, life science, biomedical imaging, etc [1, 2]. Particularly, CARS microscopy has attracted a great deal of attention as a label-free vibrational contrast imaging method that is capable of performing real-time measurements on a 3D biosample with spatial resolution in the subcellular level . For in vivo biomedical applications, however, it still remains technically challenging to develop optical fiber probes dedicated for CARS .
The CARS process involves three pulsed laser components: the pump (ωp), Stokes (ωs), and probe (ωpr). When the energy difference between the pump and Stokes components, Δω = ωp – ωs, matches to the vibration level Ωv of a molecule, then a strong anti-Stokes signal is generated at ωas = ωpr + Δω = ωpr + Ωv is generated. A typical CARS system (in which the pump laser serves also as the probe one.) employs two pulsed lasers of different wavelengths whose linewidths need to be narrow for high spectral resolution . This approach can only provide information of a single vibrational mode of the molecule at a time. In order to cover multiple vibrational modes of distinct Raman shifts Ωv, it is quite necessary to have a tunable Stokes laser (such as an optical parametric oscillator.) for which its wavelength scanning often involves a painstaking and time-consuming job, rendering real-time measurements almost impossible [6–8].
Multiplex CARS, as an alternative, provides a straightforward way to analyze many different vibrational modes in a single measurement [5, 9, 10]. Introducing a broadband light source for the Stokes laser (Δωs ≈ΔΩv) that works with a narrowband pump/probe laser (ωp = ωpr), enables a bunch of vibrational modes within a certain range of Raman shifts ΔΩv to be excited simultaneously. For a high signal-to-noise ratio (SNR) in recording multiplex CARS spectra, it is crucial that the broadband Stokes laser permits a stable and sufficient power density over a large spectral window. Interestingly, a single-pulse CARS scheme has been proposed to implement multiplex CARS using only one laser pulse [11–13]. Here, a broadband Stokes pulse from a single laser is engraved with a well-defined spectral feature for the narrowband pump/probe (e.g. spectral notch shaping, spectral phase modulation), resulting in a single versatile pulse that serves as the pump, Stokes, and probe simultaneously.
Incorporating CARS systems with fiber optics has long been pursued as well for endoscopic applications or handheld device implementations [14–16]. There have been several studies to test the fiber delivery of dual-color picosecond laser pulses for preliminary demonstrations of CARS imaging. A fiber-optic extension of CARS has been also made to construct a compact and collinear light source, ideal for broadband CARS in the molecular fingerprint region, in which a single photonic crystal fiber (PCF) is optimized to generate both the pump and Stokes lasers by exploiting the inherent nonlinear optical properties of PCFs . However, robust delivery of a custom-tailored broadband laser pulse has yet been fully addressed to realize fiber-based multiplex CARS probes [17–19].
For a fiber-optic implementation of multiplex CARS, particularly in the single-pulse scheme, it is of utmost importance to control the group velocity dispersion (GVD) and nonlinear effects experienced by a transform-limited broadband laser pulse propagating in optical fibers. This control is needed because optical nonlinearity in the fiber often deteriorates the spectral shape of the laser pulse and any GVD results in either broadening or compression of ultrashort pulses in the time domain, leading to a substantial degradation of spectral coverage and SNR measurement. Previously, we proposed the use of a fiber Bragg grating (FBG) to accomplish notch shaping of a broadband laser pulse to serve as an excitation light source of single-pulse CARS and the spectral characteristics of such FBG-filtered light source was investigated [20, 21]. We then demonstrated a single-pulse CARS experiment by using a FBG-filtered broadband laser pulse with its dispersion post-compensated for the entire setup in which a dispersion pre-compensation scheme, usually adopted in many ultrafast laser experiments, was found to be inadequate due to the adverse nonlinear effects of spectral narrowing in the fiber Bragg grating . While some limited studies have inquired into the fiber-delivered characteristics of an ultrashort laser pulse itself , establishing a dedicated fiber delivery scheme for single-pulse multiplex CARS using a spectrally shaped ultrashort laser pulse, still needs more rigorous investigations and validations.
In this paper, we present a single-pulse multiplex CARS microspectroscopy using a fiber-delivered broadband laser pulse with a narrow spectral notch. The key elements involved, especially in the fiber-optic aspect, are spectral filtering of a broadband laser pulse to form a narrow spectral dip and its transform-limited delivery to a sample through optical fibers, depending greatly on the choice of appropriate fiber components as well as a careful design to control the GVD and nonlinearity of a fiber-based system. Here, we propose a method to use an FBG that is connected directly with a large-mode-area (LMA) fiber, with the aid of a setup precisely configured for dispersion pre-compensation. The strategy for proper dispersion compensation as well as nonlinear effect suppression is revisited carefully, taking the additional LMA fiber into account. A dispersion pre-compensation scheme is found to be viable for a substantially larger negative chirp required to permit a transform-limited laser pulse at the fiber’s distal end, where the pre-chirping effectively reduces the peak intensity of the laser pulse propagating in the FBG to suppress the nonlinear effect in the fiber. The feasibility of our implementation, as investigated first by numerical simulation, is experimentally verified that it works properly as a narrowband notch filter followed by a spectral distortion-free patch cable for a broadband laser pulse. Utilizing such fiber-delivered light source suitable for single-pulse multiplex CARS, we are allowed to measure various samples having multiple Raman shifts in the fingerprint region, by recording their broadband CARS spectra with reasonable spectral stability and dynamic range. Finally, vibrational contrast imaging of spatially-dispersed poly(methyl methacrylate) microbeads is experimentally demonstrated in epi-CARS geometry where backscattered CARS signals from the raster-scanned sample are collected into a multimode optical fiber.
2. Configuration of a fiber-delivered light source for single-pulse multiplex CARS
2.1 Principle of multiplex CARS using a single notch-shaped broadband laser pulse
Unlike the typical multiplex CARS employing a narrowband picosecond laser as the pump/ probe beam and a broadband femtosecond laser as the Stokes beam in combination , our single-pulse CARS scheme uses only a single broadband pulsed laser that acts as a complete set of CARS excitation sources for itself [11–13]. The hallmark of this intriguing approach is to introduce a transform-limited broadband laser pulse which is spectrally shaped to have a narrow notch. Briefly, its working principle can be described by the induced nonlinear polarization taking an integral form as
The molecular vibration at each frequency Ω scatters the laser field E(ω) to shift in frequency by Ω, while the molecule can vibrate strongly at frequencies only around ΩR due to the resonance term in the denominator of the integrand. The nonlinear polarization, in effect, results in a form of the whole laser spectrum blue-shifted as E(ω-ΩR) with its overall intensity being proportional the Raman strength G and the laser excitation amplitude A. In Fig. 1(a) supposing that the molecule is allowed to have multiple vibrational modes within the bandwidth of a pulsed laser, broadband CARS spectral signals originating from many different Raman shifts will then overlap one another, which inevitably hampers resolving fine features of molecular vibrations. The single-pulse CARS technique, however, addresses such difficulty by adopting a simple trick that a broadband laser pulse E(ω) is engraved with a narrow (preferably, deep as well) notch at a certain frequency ωdip within its pulse bandwidth. The spectral notch profile superimposed on the excitation laser pulse plays a role of the well-defined probe beam indeed, manifesting itself as a sharp dip feature in the CARS spectrum at frequency (ωdip + ΩR) for each Raman mode while the entire CARS spectrum still retains unwanted broadband backgrounds attributed to the laser pulse bandwidth.
2.2 Fiber delivery of a notch-shaped broadband laser pulse: numerical simulation and experimental investigation
We attempt to configure a reliable experimental scheme for fiber-based filtering and delivery of a broadband laser light, being compatible with the principle of single-pulse multiplex CARS. To this purpose, it is crucial to preserve both the temporal and spectral characteristics of a notch-shaped broadband laser pulse at the distal end of the fiber used. It is well known, however, that ultrashort laser pulses transmitted in a long distance through a conventional single-mode fiber (SMF) often suffer from deterioration of their pulse characteristics, due to group velocity dispersion (GVD) and self-phase modulation (SPM) caused by optical fibers . Stretching of an ultrashort laser pulse in the time domain, caused by excessive GVD in the fiber transmission optics, can be remedied by deploying a dispersion pre-compensating setup before the fiber delivery. However, a negative chirping of the laser pulse, deliberately exerted to compensate for the positive GVD of transmission optics, might give rise to a substantial spectral narrowing of the transmitted laser pulse by SPM [24, 25]. Since accessible range of Raman shifts in the single-pulse CARS is proportional to the excitation laser bandwidth, it is absolutely necessary to circumvent the spectral narrowing occurred in fiber transmission.
Our goal is thus to achieve both the notch shaping and fiber transmission of a negatively chirped broadband laser pulse, in order to produce a desired transform-limited laser pulse without spectral narrowing at the fiber end. We suggest to employ a FBG for narrowband notch filtering and a connected LMA fiber for transmitting a near transform-limited laser pulse, which is expected to alleviate the problem of spectral narrowing effectively by a small nonlinearity of the LMA fiber itself and, in part, by the GVD pre-compensation that reduces the laser peak power.
For the simulation and experiment we used commercialized fibers. The FBG (O/E Land Inc., Canada) served as a notch filtering component to make a narrowband notch shape in the broadband laser pulse. To prevent spectral narrowing induced by SPM during notch filtering in the FBG, the incident laser pulse was temporally stretched by chirped mirror sets. The SMF (780-HP, Thorlabs) and LMA (LMA-20, core dia. 20 µm, Thorlabs), respectively, were used for the delivery and temporal compression of notch-shaped laser pulse near transform limited.
The experimental setup was constructed as presented in Fig. 2. We started with a commercial Ti:sapphire femtosecond oscillator (Micra-10, Coherent) with a spectral bandwidth of 70 nm at 800 nm central wavelength. Two chirped mirror sets (reflectivity > 99%; −175 fs2 GDD per reflection; DCMP175, Thorlabs) were used to control the large amount of group delay dispersion with high energetic efficiency. The dispersion up to −22,000 fs2 could be controlled with a net energetic efficiency of up to 80%. The negatively chirped laser pulse was focused into the FBG by the objective lens (UPlanSApo, 10X/0.40, Olympus) with a coupling efficiency of 60%. We used a commercially available fiber Bragg grating (Bragg wavelength: 790 nm; FWHM bandwidth: 1.0 nm; rejection rate > 20 dB; O/E Land Inc., Canada). After the FBG, we tested two types of optical fibers (780HP, LMA-20, Thorlabs). They were alternately connected directly with the FBG by FC/PC connector to observe the spectral change of notch-shaped laser pulses after each fiber transmission.
The spectral and temporal characteristics of the notch-shaped laser pulse were measured using a spectrometer (HR2000 + , Ocean Optics) and an interferometric autocorrelator (AA-20DD Autocorrelator, Avesta). The average power was 80 mW at the distal end of the fiber, which was necessary power to acquire the CARS signals. The initial chirp parameter of the laser pulse was controlled to produce a transform-limited pulsed laser at the exit of the fiber. The total length of the optical fiber was 30 cm (FBG: 10 cm, LMA or SMF: 20 cm). The experimental results are presented in Fig. 3(b). Due to the dispersion compensation limit through the two chirped mirror sets, the experiment was performed with a 30 cm long fiber. A longer fiber length was examined via simulation, as presented in Fig. 3(a).
A simulation was performed to compare and verify the experimental results. The conditions used in the simulations were the same as those measured in the experiment. The dispersive properties of the LMA and SMF fibers are β2_LMA = 350 fs2/cm, β2_SMF = 397 fs2/cm, β3_LMA = 282 fs3/cm, β3_SMF = 262 fs3/cm, β4_LMA = −134 fs4/cm, and β4_SMF = −67 fs4/cm [26, 27]. The nonlinear coefficients are γSMF ≈12 (W∙km)−1 and γLMA ≈1.1 (W∙km)−1. The simulation was performed under three conditions using the split step methods for the nonlinear Schrodinger equation proposed by G.P. Agrawal . The initial condition was assumed to be a Gaussian-shaped pulse with a 70 nm bandwidth and only the chirp parameter was changed. The initial chirp parameters were changed to the length of the optical fiber, and the chirp of the pulses at the distal end of the optical fiber is set to zero. We replaced the FBG by artificially creating a notch shape in the initial condition.
In the simulation and experimental results, a small amount of spectral narrowing is observed upon passing a negatively chirped laser pulse through 10 cm of FBG and 20 cm of LMA. The results are shown as the blue lines in the simulation and experiment respectively. The initial laser pulse with a 70 nm spectral bandwidth was reduced to 60 nm in both simulation and experiment. On the other hand, large amount of spectral narrowing was occurred in the FBG (10 cm) and SMF (20 cm) transmission. In Fig. 3 red lines, the spectral bandwidth of laser pulse was reduced to approximately 1/6 of its spectral bandwidth of the initial laser pulse. The experiment and simulation results reveal that the significant spectral narrowing occurred in SMF transmission. The reduced spectral bandwidth in the laser pulse hampers to access the wideband Raman modes in the single-pulse CARS scheme. On the other hand, small nonlinearity of the LMA fiber alleviate the problem of spectral narrowing effectively. The notch-shaped laser pulse delivered by LMA can be used as the incident source of single-pulse CARS for accessing the Raman modes in the molecular fingerprint region.
In addition, we investigated the case of pulsed laser delivery using longer length of LMA fiber. In Fig. 3(a) the conditions of FBG (10 cm) and LMA (200 cm) produce the similar results to the previous conditions that used the FBG (10 cm) and LMA (20 cm). It is expected that the single-pulse multiplex CARS configuration will be possible even with the longer length of LMA fiber.
The notch-shaped laser pulse used in the CARS experiment is presented in Fig. 4. In Fig. 4(a), the laser pulse with a spectral bandwidth of 60 nm and the narrowband notch shape at 790 nm was stably maintained. The unexpected spectral distortion at 780 nm was due to the characteristics of the fabricated FBG. Given that the designed notch band is sharp and deep at 790 nm, other rough spectral dips are negligible in the CARS spectrum. We also investigated the characteristics of notch-shaped laser pulse in the time domain through autocorrelation. The autocorrelation trace confirmed that the laser pulse was temporally well compressed to 30 fs. In Fig. 4(b) inset, the wings presented in the interferometric autocorrelation are attributed to the notch shape in the laser pulse generated in the spectral domain.
3. Experimental results of fiber based multiplex CARS
The experimental setup for the fiber based multiplex CARS is illustrated in Fig. 5. The Ti:S oscillator and the fiber delivery elements were identical to those in Fig. 2. The total length of the optical fiber (FBG: 10 cm, LMA: 20 cm) used in the experiment was 30 cm. To obtain transform-limited pulses at the sample position, the dispersion pre-compensation value was approximately −17000 fs2. After fiber delivery, the pulsed laser was focused on the sample using an objective lens (UPlanSApo 20X, 0.75, Olympus) with an average power of 50 mW.
Both the forward and backward scattered CARS signals were measured. A dichroic mirror (FF757-Di01 25x36, Semrock) was used to reflect the backscattered CARS signal to the multimode fiber. The backscattered CARS signal was collected into the multimode fiber (M71L01 core diameter of 1000 µm 0.48 NA, Thorlabs) and delivered to a spectrograph (Triax 320 Jobin Yvon) and EMCCD (Newton EMCCD DU970N-BV, Andor). Only the CARS components were collected, and the notch-shaped laser pulse was rejected by the bandpass filter (FF01-708/75-25, Semrock). We measured liquid samples with a strong Raman peak in the molecular fingerprint region. The exposure time for the forward CARS signals was 50 ms.
Figure 6 presents experimental results of various samples. For the samples with a vibrational mode in the molecular fingerprint region, a dip shape is observed at specific spectral locations that corresponded to the vibrational modes. For acetone, the notch location is 790 nm (12658 cm−1) with a dip at 743.5 nm (13449 cm−1), indicating that the Raman mode occurs at 790 cm−1. Acetone exhibits a strong Raman peak at 790 cm−1. Other samples have also a dip feature at different wavelength.
Vibrational spectra from various samples in the fingerprint region are presented in Fig. 6(b). All CARS spectra were normalized by the nonresonant CARS spectrum from the water sample, which has no Raman lines in our detectable range. For each sample, the normalized CARS signal is consistent with previously reported Raman-active mode values . The detectable range and the spectral resolution of the CARS signal are determined by the spectral bandwidth and notch bandwidth of the laser pulse. In our experiment, the detectable range was from 630 cm−1 to 1040 cm−1 and the spectral resolution was 20 cm−1.
Finally, vibrational contrast imaging of spatially-dispersred poly(methyl methacrylate) (PMMA) (SUNPMMA-S150, Sunjin Chemical Ltd., South Korea) microbeads is experimentally demonstrated in epi-CARS geometry. In Fig. 5, we constructed a sample stage incorporating two motorized stages (MT1-Z8, Thorlabs) for raster scanning along the X and Y axes. At each point, the backscattered CARS signals were obtained after an integration time of 1 s.
The PMMA beads used in the experiment was confirmed to have two Raman modes (810 cm−1 and 975 cm−1) within our detectable CARS range by a Raman spectrometer (LabRAM HR Evolution, Horiba, France). Figure 7(a) presents the backscattered CARS spectrum of poly(methyl methacrylate) beads embedded in agarose gel. The spectral dip features in the backscattered CARS spectrum is observed at 742.5 nm and 733.5 nm. The dip features at 742.5 nm (13449 cm−1) and 733.5 nm (13633 cm−1) indicate that the Raman modes were detected at 810 cm−1 and 975 cm−1 in the PMMA which is consistent with previous experiment with the Raman spectrometer. The red line in Fig. 7(a), which represents the nonresonant CARS spectrum of agarose gel, exhibits no Raman lines in the detected range.
As shown in Fig. 7(c), we acquired a raster-scanned CARS image of 5~20 µm in diameter poly(methyl methacrylate) beads embedded in agarose gel. For comparison, a conventional bright field image of sample is presented in Fig. 7(b). A relatively poor spatial resolution and distortion apparent in the CARS image for microbeads, as compared with its bright-field counterpart, can be attributed to several technical shortcomings of our present experimental setup. They include (i) a coarse step size (1 μm) of the raster scan allowing its spatial resolution no better than a few micrometers, (ii) uncorrected chromatic aberration of the microscope objective presumably leading to an excessive blur of the focal spot, (iii) a drift of the focal plane during a whole CARS image acquisition for microbeads distributed with different heights, (iv) mechanical imperfection of the motorized sample stage suffering some nonlinear movement and backlash, etc. However, there is still a lot of room for future improvement regarding the spatial resolution of single-pulse CARS imaging. Based on the experimental results, we verified that the fiber-delivered notch-shaped laser pulses can be used as an excitation light source for the single-pulse multiplex CARS microspectroscopy.
We demonstrated a fiber-delivered broadband laser pulse for multiplex CARS microspectroscopy in the molecular fingerprint region. We demonstrate that a FBG can serve as a narrowband notch filter for broadband femtosecond laser pulse and a LMA fiber can deliver the negatively chirped laser pulse to the sample without severe spectral distortion. The notch-shaped laser pulse using FBG and LMA for single-pulse multiplex CARS was verified by simulation and experiments. Using the notch-shaped laser pulse delivered by optical fibers, we obtained the CARS spectra of various samples of molecular vibration in the fingerprint region. Furthermore, CARS imaging of poly(methyl methacrylate) bead samples was successfully performed under epi-CARS geometry in which backscattered CARS signals. Since high power optical fiber based mode-locked lasers have been developed, the compatibility of single-pulse CARS scheme with fiber optics, verified in this study, implies a potential for a realization of compact all-fiber CARS spectroscopic imaging system.
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