3656 OPTICS LETTERS / Vol. 31, No. 24 / December 15, 2006

Dual-pump coherent anti-Stokes–Raman scatteringmicroscopy

Ondrej BurkackyDepartment Chemie, Ludwig-Maximilian University, Butenandstraße 11, 81377 Munich, Germany

Andreas ZumbuschDepartment Chemie, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany

Christian Brackmann and Annika EnejderChalmers University of Technology, Fysikgränd 3, S-412 96 Gothenburg, Sweden

Received August 22, 2006; revised September 28, 2006; accepted September 28, 2006;posted October 2, 2006 (Doc. ID 74343); published November 22, 2006

We introduce dual-pump coherent anti-Stokes–Raman scattering (dual-CARS) microscopy. This new tech-nique permits simultaneous imaging of two species characterized by different molecular vibrations, as wellas the removal of nonresonant background. This is achieved by using three synchronized laser pulses prob-ing two different vibrations. We demonstrate the virtues of the method by imaging a mixture of nondeuter-ated and deuterated lipids, clearly distinguishing the individual components and their organization in themixed arrangement. Further, dual-CARS images of lipid stores in living Caenorhabditis elegans nematodesshow that the suppression of the nonresonant background results in significantly enhanced image contrast.© 2006 Optical Society of America

OCIS codes: 110.0180, 180.5810, 190.4380, 300.6230.

Since the introduction of coherent anti-Stokes–Raman scattering (CARS) microscopy with collinearexcitation,1 the technique has developed to become avaluable tool for investigations of unlabeled speci-mens within the biosciences and material sciences.2–4

CARS is a four-wave mixing process, in which threephotons interact with the molecules in the sample,generating an anti-Stokes photon. Photons providedby a pump pulse and a Stokes pulse of frequencies �pand �s, respectively, generate an anti-Stokes pulseemitted at the frequency �CARS=2�p−�s. The inten-sity of the generated CARS signal can be written as

ICARS��� � ��CARS�3� ����2Ip

2Is = ��r�3���� + �nr

�3��2Ip2Is, �1�

where Ip and Is are the intensities of the pump andStokes beams, respectively, and � is the frequencydifference �p−�s. The third-order nonlinear suscepti-bility �CARS

�3� can be decomposed into a resonant �r�contribution, accounting for the molecular vibration,and a general, essentially frequency-independent5

nonresonant (nr) contribution. The latter makes itdifficult to discern structures of weak Raman scatter-ers, leading to a significant reduction of the micros-copy image contrast. Several methods have been de-veloped for suppression of the nonresonantbackground. Among these are the use of polarizationschemes,6 time-resolved methods,7 and epide-tections,8 all of which are also accompanied by a se-vere attenuation of the resonant CARS signal. Themultiplex CARS method9–11 allows detection over abroad vibrational region, allowing the nonresonantbackground to be measured and compensated for.However, it requires long integration times and usu-ally precise temporal synchronization of a femtosec-

ond and a picosecond laser oscillator. Heterodyne

0146-9592/06/243656-3/$15.00 ©

CARS12 eliminates the nonresonant background byusing interferrometric microscopy. This requires anadvanced interferometer setup combined with thelock-in detection technique, which may limit the dataacquisition rate. Recently, frequency-modulatedCARS13 has been demonstrated, using alternatingpump beams tuned on and off a vibrational resonancecombined with lock-in detection. However, themethod does not allow for the excitation of two vibra-tional resonances simultaneously.

Here, we introduce dual-CARS microscopy with anexcitation scheme consisting of three synchronizedlaser pulses in a dual-pump configuration.14 This al-lows the simultaneous collection of images at theresonant vibration and of the nonresonant back-ground. CARS microscopy images can then be ex-tracted free from the nonresonant background andpulse jittering effects, improving the sensitivity ofthe technique. Moreover, the dual-pump scheme al-lows the unique ability to probe two arbitrarily cho-sen molecular vibrations simultaneously. Whereasmultiple vibrations can also be probed by means ofmultiplex CARS9–11 and chirped excitation pulses,15

these techniques require long integration times, andthe vibrational range covered is limited by the band-width ��300 cm−1� of the excitation pulses. Dual-CARS microscopy offers imaging of fast interactionmechanisms and colocalization of species with vastlydifferent molecular vibrations, opening the way for awide range of interesting cell-biological and material-science studies.

The experimental setup (Fig. 1) is based on a lasersystem consisting of a Nd:vanadate laser (HighQ,7 ps, 1064 nm, 76 MHz repetition rate, linewidthtypically 5 cm−1) pumping two intracavity-doubledoptical parametric oscillators (APE Levante), and an

2006 Optical Society of America

December 15, 2006 / Vol. 31, No. 24 / OPTICS LETTERS 3657

inverted microscope with a mirror scanning unit (Ni-kon Eclipse TE2000-E and Nikon C1). The wave-lengths of the optical parametric oscillators (OPOs)can be tuned individually between 786–845 nm and852–920 nm, respectively. The output beams of theOPOs and a fraction of the 1064 nm beam are com-bined and directed into the scanning unit of the mi-croscope. Temporal overlap between the laser pulsesat the sample is achieved by two adjustable beam de-lay lines. A water immersion objective (Nikon APO60�, NA 1.2) is used to focus the beams onto thesample.

The sample is simultaneously illuminated with theoutput from the two OPOs and the Stokes beam at1064 nm (the typical laser power at the sample is30 mW per beam), probing two Raman active vibra-tions simultaneously. Two corresponding CARS sig-nals and a mixing component of frequency �aS3=�p1+�p2−�s without chemical specificity are emitted.The CARS signals, copropagating with the laserbeams, are collected by an aspherical lens, separatedby a short-pass beam splitter, and directed to twosingle-photon counting photomultipliers (Hama-matsu). The detectors are equipped with differentsets of bandpass filters, which suppress the laserwavelengths and the mixing signal while transmit-ting the CARS signals.

The intensities in the two channels are related toeach other by probing a completely nonresonantsample, in our case a glass coverslip. As the nonreso-nant susceptibility contribution �nr

�3� is essentially in-dependent of the excitation wavelengths, the de-tected signals can be considered the same for bothchannels. With pump intensities Ip1 and Ip2, andchannel detection efficiencies �1 and �2, the calibra-tion ratio I1/2= ��1Ip1

2 � / ��2Ip22 � is obtained. This calibra-

tion measurement is required when the dual-CARSconcept is used to extract a background-free CARSimage from simultaneously recorded resonant ��1�

Fig. 1. (Color online) Scheme of the experimental setupfor dual-CARS microscopy. The three excitation pulses aregenerated by a Nd:V pump laser and two OPOs, over-lapped in time and space, and coupled into a laser scanningmicroscope. An objective focuses the beams in the sample,and the emitted CARS signal is collected by an asphericallens. A short-pass beam splitter separates the dual-CARSsignal into its two components for detection by separatephotomultiplier tubes. BS, beam splitter; DM, dichroic mir-ror; SP, short-pass beam splitter; R, retroreflector.

and nonresonant ��2� images of the target sample.

The nonresonant contribution in channel 1 can thenbe calculated from the intensity measured in the non-resonant channel 2 by using the calibration ratioI1/2 :ICARS��2�I1/2=�1 ��nr

�3��2Ip12 Is. The pure resonant im-

age in channel 1 can then be obtained as

ICARS, resonant = ICARS��1� − ICARS��2�I1/2 = �1��r�3��2Ip1

2 Is,

�2�

with the assumption that Re��r�3�� is insignificant

relative to Im��r�3�� at resonance.

Dual-CARS measurements were made on a mix-ture of crystals of deuterated and nondeuterated tri-palmitin, and on living Caenorhabditis elegansnematodes of a feeding deficient mutant. The formersample illustrates the ability to visualize two molecu-lar vibrations simultaneously (Fig. 2). The CH2 sym-metric stretch vibration at 2845 cm−1, detected at663 nm ��1�, is monitored in one channel [Fig. 2(a)],while the C–D (carbon deuterium) vibration at2115 cm−1 is simultaneously detected at 734 nm ��2�in the second channel [Fig. 2(b)]. High contrast is ob-tained in all images without any cross talk betweenthe detection channels. The two-channel overlay pic-ture shown in Fig. 2(c) was formed by merging the in-dividual CARS images coded in different colors.These images clearly demonstrate the possibility toimage two species simultaneously with high chemicalselectivity, allowing for the interaction and colocal-ization studies.

A dual-CARS image of a living biological specimenis shown in Fig. 3. It demonstrates that the conceptof dual-CARS microscopy efficiently can be used toeliminate the nonresonant background for improvedsensitivity. We probed living C. elegans nematodes,having the lasers tuned to the same wavelengths asfor crystal measurements. As there are no C–D bondsnaturally occurring in the nematode, the signal fromthe C–D channel can be entirely attributed to thenonresonant background. Figures 3(a) and 3(c) showthe raw data acquired in each channel. Figure 3(a)includes both resonant and nonresonant signal con-tributions, whereas the isolated nonresonant back-ground contribution is shown in Fig. 3(c) (compen-sated for detection efficiencies and laser intensities

Fig. 2. (Color online) Dual-CARS microscopy images of amixture of deuterated and nondeuterated tripalmitin. Si-multaneous measurements were made with the laserstuned to (a) the CH2 vibration at 2845 cm−1, and (b) theC–D vibration at 2115 cm−1. Image (c) is the correspondingoverlay image, where the red (dark) color represents theCH2 vibration, and the yellow (bright) color represents theC–D vibration. The organization of the different crystalshere is clearly visualized. The image dimensions are 100

2

�100 �m , and the integration times were 60 s each.

3658 OPTICS LETTERS / Vol. 31, No. 24 / December 15, 2006

using the calibration ratio). Employing Eq. (2) on thedata of Figs. 3(a) and 3(c), the background-free imageshown in Fig. 3(e) can be extracted. In this image,contrast arises solely from the CH2 vibrational mode.The lipid stores inside the nematode can clearly bedistinguished here from the background, despitetheir depletion and small size due to the feeding de-ficiency of the species studied. The intensity profilesalong the white lines in Figs. 3(a), 3(c), and 3(e), areshown in Figs. 3(b), 3(d), and 3(f), respectively. Com-paring Figs. 3(b) and 3(f), we conclude that thesignal-to-background ratio has improved by a factorof 3. It is worth mentioning here that compensationfor the nonresonant background using dual-CARS is

Fig. 3. (Color online) Elimination of the nonresonantbackground by means of dual-CARS microscopy, exempli-fied by CARS images of a living C. elegans nematode. Im-age (a) was recorded at the symmetric CH2 stretch vibra-tion �2845 cm−1� and image (c) at the C–D vibration�2115 cm−1�. Image (c) visualizes the nonresonant back-ground and has the same color scale as image (a). Image (e)is the pure resonant image calculated from Eq. (2), clearlyvisualizing the small and depleted lipid stores in the feed-ing deficient species. (b), (d), and (f) show normalized inten-sity profiles along the white lines in the corresponding im-ages (a), (c), and (e). Profiles (b) and (f) show animprovement in the signal-to-background ratio by a factorof 3 for the processed image (e). The image dimensions are50 �m�50 �m, and the integration times were 20 s each.

generally applicable to the imaging of biologicalsamples, since these have a resonance-free spectralwindow of some hundred wave numbers width cen-tered at 2400 cm−1. In summary, dual-CARS micros-copy permits the simultaneous visualization of twomolecular vibrations, allowing studies of interactionphenomena and colocalization of cell-biologically rel-evant molecules. Dual-CARS microscopy also enablesthe collection of CARS microscopy images free fromthe nonresonant background, significantly improvingthe sensitivity and image contrast. This is essentialfor biological applications, here allowing for the visu-alization of depleted lipid stores in feeding deficientC. elegans nematodes.

The C. elegans samples were kindly provided byClaes Axäng, the Department of Molecular Biotech-nology. valuable discussions with Thomas Hellererare gratefully acknowledged. The experimental setupwas funded by the postgenomic research and technol-ogy program in southwest Sweden (SweGene). O.Burkacky acknowledges the receipt of a travel allow-ance from the Boehringer Ingelheim Fonds. C. Brack-mann is supported by the Carl Trygger Foundationand A. Enejder by the Swedish Research Council. A.Zumbusch’s e-mail address is Andreas.Zumbusch@uni-konstanz.de.

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