Chemical Imaging With Frequency Modulation Coherent Anti-Stokes Raman .

Chemical Imaging with FM-CARS MicroscopyJ. Phys. Chem. B, Vol. 114, No. 50, 2010 16873Figure 2. Comparison of CARS methods. (a and b) Temporal profilesof pump [EP(t)], Stokes [ES(t)] and excitation [A(t)] fields in the narrow(a) and spectral focusing (b) CARS methods, respectively. (c) Laserspectra used in the simulations of part d. The pulse energies are identicalin both cases. (d) Simulated CARS spectra of the narrow (black) andspectral focusing (red) methods. Note the similar signal strengths inthe center frequency region. Traces in parts a and b are verticallydisplaced for clarity.time domain (with the same GVD) and some parts of that pulsewill not be used for CARS signal generation.Next, we compare the efficiencies of signal generation bythe spectral focusing and narrow CARS methods. Figure 2a,bshows EP(t), ES(t), and A(t) in the narrow and spectral focusingmethods, respectively. We apply an appropriate GVD to thebroad-band pulses in Figure 2b such that both cases haveidentical pulse durations. Note that the phases of A(t) in Figure2a,b are different due to the condition of the simulation, whichdoes not affect the magnitudes of the generated CARS signals.As one can see in Figure 2a,b, amplitudes and shapes of thefield envelope of A(t) are identical in both methods.26 This canbe understood easily by the following consideration. At anygiven time point, the amplitudes of laser fields in both casesare identical, thus its beating components should be of equalmagnitudes.CARS signals are generated when the pump pulse interactswith the vibrational excitation again. Figure 2d shows thesimulated CARS signals by each method. The CARS signals iscalculated by8SCARS(ω) ) dω′ χR(ω′)EP(ω - ω′) dω′′ EP(ω′′ω′)ES(ω′′) 2(5)where SCARS(ω) is the intensity of CARS signal photons at thesignal frequency of ω, χR(ω′) is the third-order vibrationalsusceptibility of the sample at the vibrational frequency of ω′.The laser spectra of the pump and Stokes pulses used in thissimulation are shown in Figure 2c. The bandwidths (fwhm) ofthe narrow and broad CARS pulses are 10 and 450 cm-1,respectively. We apply an appropriate GVD to the broad-bandpulses to match the pulse durations to be identical in both cases.The pulse energies ( E(ω) 2 dω) are also set to be equal. Anartificial sample is assumed to possess five vibrational peakswith identical peak intensities. In the case of the narrow CARSmethod, we scan the frequency of the Stokes pulse to generatea CARS spectrum. In the spectral focusing method, we changethe time delay by adding a group delay (i.e., linear spectralphase) to the Stokes pulse. As one can see in Figure 2d, it isclear that both methods have the same signal generationefficiencies as long as the pulse durations are identical. Thespectral focusing method generates weaker CARS signals at thelower and higher vibrational frequency regions than the narrowmethod does. The signals decrease since there are feweravailable pairs of the pump and Stokes pulses to excite vibrationsat these frequency regions.7-9 However, this limitation can beovercome with laser pulses with a broader bandwidth.7 In thiswork, we use laser pulses with a bandwidth of 1800 cm-1 andobtain CARS signals over 800-1700 cm-1 that covers most ofthe fingerprint region.In CARS microscopy and spectroscopy, the nonresonantbackground always accompanies the vibrational CARS signal.1It not only adds up as a smooth background but also interfereswith CARS signals to distort its spectral shape.4 In CARSimaging at a single vibrational frequency, the nonresonantbackground can mislead the interpretation of local chemicalstructures.27,28 One method to eliminate the effect of thisproblematic background is frequency modulation (FM).13-16Since the nonresonant background originates from the electronicresponse of the sample, its magnitude is insensitive to thevibrational excitation frequency (i.e., Ω in eq 4). If one canobtain the CARS signals at different Ω values, the nonresonantbackground can be subtracted. The FM-CARS technique hasbeen shown to be very effective in eliminating the nonresonantbackground.13 In the previously demonstrated narrow FM-CARSmicroscopy methods, one needs either two different pump lasersthat are switched by a Pockels cell13 or a sophisticated opticalparametric oscillator (OPO) that can modulate the frequencyof the output pulses.16 In the spectral focusing CARS, FM canbe realized in a much simpler way. Since the excitationfrequency can be changed by time delay, FM-CARS can beimplemented by switching optical paths, not the actual frequencyof laser pulses.14,15III. Experimental SectionIn this work, we use broad-band pulses from a single cavitydumping Ti:sapphire oscillator laser (Cascade, KM Lasers) togenerate CARS signals. The spectrum of our laser is shown inthe inset of Figure 3. The output power of this laser is 40 nJ ata repetition rate of 2 MHz. Figure 3 shows our experimentalsetup for FM-CARS microscopy. The laser beam is split by a50:50 beam splitter (CVI) and each arm serves as the pumpand Stokes pulses. In the pump arm, a Pockels cell (ConOptics,model 350-50C) is used to switch the polarization direction ofincoming pulses. Depending on the on-off state of the Pockelscell, the pump pulses travel along one of the two different paths(pump-1 and pump-2 shown in Figure 3) separated by apolarizing beam splitter. These two pump pathlengths are setto be slightly different. The returning pump pulses pass thePockels cell again to recover the original polarization states.We modulate the Pockels cell with a 100 kHz square drivingvoltage waveform, which is synchronized to the laser pulse train.In the Stokes arm, we insert a 2.6 cm long SF57 glass rod(Casix) to match the amount of GVD in the pump and Stokespulses. The mirror in the Stokes arm is positioned on acomputer-controlled translation stage (M.405-CG, PI), whichcan be delayed at a maximum speed of 1 mm/s. The pump and

16874J. Phys. Chem. B, Vol. 114, No. 50, 2010Chen et al.Figure 3. Experimental setup for FM-CARS microscopy. BS, 50:50beamsplitter; PC, Pockels cell; PBS, polarizing beam splitter; G, SF57glass; SM, galvo scanning mirror; L1, scan lens; L2, tube lens; OL,microscope objective lens; S, sample; F1, long wavelength pass filter;F2, short wavelength pass filters; Lock-in, lock-in amplifier. (Inset)Laser spectrum.Stokes pulses are recombined by the beam splitter and travelcollinearly afterward. A sharp-edge long wave pass filter(740AELP, Omega Optical) removes laser frequency components shorter than 740 nm. The laser pulses are then sent to ahomemade microscope. Our microscope uses a galvo scanner(GVSM002, Thorlabs) to raster scan the laser focal spot on thesample plane. The laser pulses are focused into a sample by a1.2 NA water immersion objective lens (Olympus) and CARSsignals are collected by a 0.65 NA air objective lens (Olympus).The laser pulses are removed by two sharp-edge short wavepass filters (710 ASP, Omega Optical), and CARS signals aremeasured with a PMT (Hamamatsu, H9656-20). The electricaloutput of the PMT is fed into a lock-in amplifier (SR830, SRS)referenced to the driving voltage waveform of the Pockels cell.The maximum speed of acquiring an image of 200 200 pixelsis around 1 frame/s (25 µs pixel dwell time). This speed ismostly due to the modulation speed of our Pockels cell (100kHz) and the time constant of the lock-in amplifier. All thespectra shown in this work are taken under the microscope setupin 0.5 s, which is limited by the speed of our current translationalstage. Most of CARS measurements in this work are performedwith the pump and Stokes powers of 12.0 and 11.0 mW,respectively. These powers are measured before the 1.2 NAobjective lens.The fatty acids used in this work are stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidonic (20:4), eicosapentaenoic (EPA) (20:5), and docosahexaenoic (DHA) (22:6)acids. These oils are purchased from Sigma-Aldrich and usedwithout further purification. Oil droplets are prepared in thefollowing way:29 0.05 g of each oil is mixed with 0.05 g ofTriton X-100 (Sigma-Aldrich) and stirred for 5 min, and 6 mLof water is added to the mixture, which is stirred further for2 h. Separately prepared olive and fish oil droplets are mixedafterward. Multilamellar vesicles (MLV) are prepared fromDPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, AvantiPolar Lipids). DPPC powder dissolved in a chloroform/methanol(9:1 vol/vol) solvent is evaporated under vacuum for 6 h andprehydrated with nitrogen saturated with water vapor for 15min. A HEPES buffer (10 mM, pH 7.0) is then added to thelipid film at 37 C and lipid forms MLVs.For skin imaging, the ear from a white, wild-type mouse isused immediately after sacrificing. A thin sliced skin piece( 300 µm thick) is immersed in DMSO solvent for 20 minFigure 4. Effect of GVD on spectral focusing CARS. (a) Experimentalsetup with a pulse shaper. GR, grating; SLM, spatial light modulator;S, sample; F, short wave pass filter. (b) Phase mask for spectral focusingCARS spectroscopy. The center frequency of the Stokes phase maskis shifted to delay the Stokes pulse. (c) Experimentally measured CARSspectra of toluene by pulses with different durations. (d) Normalizedtraces of part c.and placed between two coverslips. For plant cell wall imaging,a thin piece of a corn leaf is used.IV. ResultsEffect of GVD on Spectral Focusing CARS. We first studythe effect of the amount of GVD on both spectral resolutionand intensity of CARS signals with phase-controlled pulses. Theexperimental setup is shown in Figure 4a. Note that we use thesame laser for both FM-CARS (Figure 3) and the phase-controlCARS (Figure 4a) experiments. Detailed explanation of ourpulse shaper can be found in our earlier publication.7 Themicroscope, filter, and detector are identical to those used inthe FM-CARS setup. The laser pulse is first compressed to betransform-limited at the focus of microscope sample positionby the homodyne SPIDER method that we developed recently.30Then, we select the pump and Stokes pulses from a single broadband pulse by applying a phase mask of Figure 4b. The laserfrequency region from 740 to 800 nm is used as the pump andremaining region as the Stokes (Figure 4b). By controlling theslope of the quadratic phase masks (GVD) of Figure 4b, wechange pulse durations quantitatively. The CARS signal ismeasured by a PMT while scanning the time delay of the Stokespulse by shifting the center frequency of its phase mask (Figure4b). Note that this phase scan corresponds to the time delay ofthe Stokes pulse. Figure 4c shows the measured spectral focusingCARS spectra of toluene by pulses of 0.7-4.5 ps durations.Shorter pulses generate stronger CARS signals. Their vibrationalpeaks, however, are broad and more distorted (Figure 4c). Figure4d shows the normalized CARS spectra of those in Figure 4c.One can clearly see that the spectral resolution dramaticallyimproves as the pulse is stretched.20 However, it cannot exceedthe natural line width of the molecular vibration. While theCARS signals from 3 and 4.5 ps pulses show almost identicalvibrational linewidths (Figure 4d), the signal strength with 4.5pulses is only half of the one with 3 ps pulses (Figure 4c).FM-CARS Spectroscopy. Figure 5a shows spectral focusingCARS spectra of cyclohexane obtained in our FM-CARS setup

Chemical Imaging with FM-CARS MicroscopyJ. Phys. Chem. B, Vol. 114, No. 50, 2010 16875Figure 5. FM-CARS spectroscopy. (a) Measured CARS signals ofcyclohexane by the pump-1 (black) and pump-2 (gray) over time delay.The other pump beam is blocked for these measurements. (b) FMCARS signal measured with both the pump-1 and pump-2 pulses. Seethe text for details.with only one of the pump-1 and pump-2 beams. The otherpump beam is blocked to obtain these spectra. As emphasizedearlier, the key requirement of the spectral focusing CARSmethod is the identical GVD of the pump and Stokes pulses.18In our experiments, the blue part of the laser pulse acts as thepump and the red part as the Stokes to excite CARS signals atthe fingerprint region. We characterize the balance of GVD inthe pump and Stokes pulses by the spectral resolution of theCARS spectrum from cyclohexane. We find that the pulseduration of our pump pulse is 1 ps at the sample position withoutan additional glass in our FM-CARS setup. Note that the Pockelscell, polarizing beam splitter, objective lens, and other opticsadd a significant GVD on our laser pulses. We experimentallyfind that inserting an additional 2.6 cm of a SF57 glass(refractive index of 1.82) in the Stokes arm balances the GVDof pump and Stokes pulses.As one can see in Figure 5a, the CARS signals are distortedby the nonresonant background, which is well-understood inthe CARS community.1 With the presence of the nonresonantbackground, the measured CARS signal isS(ω) PR(ω) PNR(ω) 2 ) PR(ω) 2 PNR(ω)2 2PNR(ω) Re[PR(ω)] (6)where S(ω) is the measured signal at the frequency of ω, andPR(ω) and PNR(ω) are the transient polarizations for the resonantCARS signals and nonresonant backgrounds at ω, respectively.We find that most fingerprint CARS peaks from nonaromaticorganic samples fall in the so-called heterodyne limit [PR(ω), PNR(ω)] in our method. In this regime, the measured signalsbecome7S(ω) PNR(ω)2 2PNR(ω) Re[PR(ω)](7)Note that the measured signal [S(ω)] is the CARS signalmultiplied by the nonresonant background {2PNR(ω) Re[PR(ω)]}on top of the nonresonant background [PNR(ω)2]. In other words,the CARS signal is amplified by the nonresonant background,which is referred to as heterodyne amplification.13,31 In thisFigure 6. (a) FM-CARS spectra of stearic (SA, 18:0), oleic (OA, 18:1), and linoleic (LA, 18:2) acids and EPA (20:5). (b) SpontaneousRaman spectra of the same fatty acids. Each trace is vertically displacedfor clarity. CARS and Raman spectra for OA and LA are scaled twice.regime, measured CARS signals also become linearly proportional to sample concentration.13,29When both pump pulses are unblocked, the lock-in amplifiermeasures the difference between these two CARS signals, andthe spectral shape of its output looks close to the shape ofspontaneous Raman scattering (Figure 5b). Since we can monitorthe entire CARS spectrum in real time by fast scanning of theStokes pulse delay, we adjust the mirror position of the pump-2to maximize the spectral resolution of FM-CARS signals withminimum loss of its peak intensity.14 The entire vibrationalspectrum over 800-1700 cm-1 is measured by scanning thetime delay of the Stokes pulses. Despite of the existence of smalldips around the peaks, the FM-CARS spectrum reveals narrowindividual vibrational peaks and can be directly correlated tothe spontaneous Raman spectrum. We convert time delay intovibrational frequency with the known Raman peak positions ofcyclohexane. An excellent linear correlation between the timedelay and vibrational frequency is found. Due to the mismatchedhigh-order chirps between the pump and Stokes pulses, wecannot maximize the spectral resolution over the entire spectralrange. One can notice that the 1444 cm-1 peak of cyclohexanein Figure 5b is slightly broader than other peaks. As one cansee in Figure 5b, however, we are able to achieve good spectralresolution over 900-1500 cm-1. The fwhm of the 1028 cm-1peak in Figure 5b is around 20 cm-1.Quantitative Chemical Identification of Fatty Acids. Figure6 shows the FM-CARS and spontaneous Raman spectra ofstearic (18:0), oleic (18:1), and linoleic (18:2) acids and EPA(20:5). Since these spectra contain rich information frommultiple characteristic vibration peaks such as olefinic CdCand methylene CH2 deformations, it is possible to identifymolecular structures by quantitative spectral analysis.32,33 Ramanspectra in Figure 6b are measured under the same microscopewith a homemade confocal Raman microscope that consists ofa 785 nm continuous wave laser and a spectrometer (Holospecf/1.8, Kaiser Optical System) coupled with a cooled CCD (iDusDU420A, Andor). We observe all the major Raman peaks inthe FM-CARS spectra. Vibrational peak positions of FM-CARSspectra are matched with those of Raman spectra within 10cm-1. The relative peak intensities in the FM-CARS andspontaneous Raman spectra, however, show some degree ofdifferences. This is mainly due to the limited bandwidth of ourlaser pulses, as explained in section II.7 We also notice that thepeak ratio in FM-CARS is significantly different from that in

16876J. Phys. Chem. B, Vol. 114, No. 50, 2010Figure 7. The intensity ratios (a) I1260/I1300 and (b) I1640/I1460 versusthe unsaturation level of fatty acids. The unsaturation level is calculatedby the number of CdC bonds per CH2. In the plot also shown are theconventional notations (X:Y) for fatty acids. The vertical error barsrepresent the standard deviations over 10 independent measurements.Raman when the peaks are very close to each other. Oneexample is the 1260 and 1300 cm-1 peaks in Figure 6a. Weemphasize, however, that the relative peak ratio in FM-CARSspectra of the same sample is consistent from different measurements. Thus, we can identify the sample by the vibrational peakanalysis once we know the FM-CARS spectrum of the puresample.Figure 7 shows examples of quantitative CARS spectralanalysis. Here, we measure the FM-CARS spectra of a seriesof fatty acids that have different unsaturation levels. Note thatthe notation X:Y here corresponds to the numbers of carbonatoms (X) and double bonds (Y) in the acyl chain, respectively.In the fingerprint region, there are several distinct features inRaman spectra of fatty acids (Figure 6b). In the followingdiscussion, we round the vibrational peak positions to the nearesttens.C-C Stretching Region (1000-1200 cm-1). This regioncontains peaks due to the gauche and trans conformations ofthe acyl chains.32,34,35 Liquid fatty acids show weak featurelessbands while solid ones have distinct triplet peaks. This featureis a great indicator of the thermodynamic phase of fatty acidsand phospholipids.Intensity Ratio of 1260 and 1300 cm-1 Peaks. In theliterature, these peaks are assigned to in-plane olefinic hydrogenbending (1260 cm-1) and methylene twisting deformation (1300cm-1).32,34 The 1260 and 1300 cm-1 peak intensities increasewith the numbers of CdC double bonds and C-C single bonds,respectively.36 Note that the relative intensity of 1260 cm-1 peakper a CdC double bond is significantly higher than that of

The first step in third-order coherent Raman processes is to excite coherent molecular vibrations by beating frequency between two pulses (pump and Stokes pulses). The frequency of excited coherent vibrations corresponds to the frequency difference between the pump and Stokes pulses.8,22,24 Let us consider the nonlinear vibrational excitation .