March 15, 2009 / Vol. 34, No. 6 / OPTICS LETTERS 773

Comparing coherent and spontaneous Ramanscattering under biological imaging conditions

Meng Cui, Brandon R. Bachler, and Jennifer P. Ogilvie*Department of Physics and Biophysics, University of Michigan, Ann Arbor, Michigan 48109, USA

*Corresponding author: jogilvie@umich.edu

Received December 8, 2008; revised January 23, 2009; accepted January 23, 2009;posted February 3, 2009 (Doc. ID 104936); published March 9, 2009

We compare imaging based on coherent and spontaneous Raman scattering (SpRS) under conditions rel-evant for biological imaging. Using a broadband laser source, we perform spectral domain imaging of poly-styrene beads using coherent Stokes Raman scattering and SpRS and find comparable signal levels. Shortinteraction lengths, low molecule number, and low incident power all reduce the advantages available withcoherent Raman methods. We present calculations to support our measurements. © 2009 Optical Society ofAmerica

OCIS codes: 180.4315, 180.5655, 300.6230, 300.6450.

The promise of endogenous, molecularly specific con-trast has made coherent anti-Stokes Raman scatter-ing (CARS) microscopy an active field of research[1–5]. Compared to spontaneous Raman scattering(SpRS), CARS spectroscopy has been shown to pro-vide orders of magnitude higher signals under appro-priate experimental conditions [6]. For biological im-aging the appeal of CARS is multifold; higher signalsmay permit faster acquisition times and the use oflower incident power, reducing sample damage. Themultiphoton nature of CARS facilitates 3D imagingdue to the inherent localization of the signal. Thehigher frequency of CARS signals permits easy sepa-ration from fluorescence background. A significantdrawback of CARS compared to SpRS is its more-difficult implementation and the presence of a non-resonant (NR) background signal that distorts lineshapes, reducing chemical contrast. Following itsimplementation with near-IR lasers [3], the develop-ment and applications of CARS microscopy havesteadily increased. However, there has been a lack ofsystematic comparison in the literature between co-herent and spontaneous Raman signals under theconditions appropriate for biological imaging. In thisLetter we make this comparison and find that oftenthe signal strengths are comparable, contrary to thecommon assertion in the microscopy literature thatCARS signals are much stronger than SpRS signals[1,2,4].

Early work comparing the relative sensitivity of co-herent and spontaneous Raman techniques pointsout that the method of choice depends heavily on theapplication; CARS is favored when high incidentpower is used to probe large numbers of molecules[7]. Recent studies using time-delay methods to sup-press the NR background have compared coherentStokes Raman scattering (CSRS) and SpRS with alow-repetition-rate laser in condensed phase and mi-crospectroscopy studies, finding that CSRS providesimprovements of 100–105 with nanojoule–microjoulepulse energies [8,9]. Biological applications of nonlin-ear microscopy typically employ high-repetition-ratelasers and lower pulse energies to permit rapid imag-

ing and preserve sample integrity. Photodamage

0146-9592/09/060773-3/$15.00 ©

studies suggest that with near-IR 80 MHz excitation,powers of 2.5–7.3 mW cause loss of cell reproductionability for pulses ranging from 150 fs–2.2 ps in dura-tion [10,11].

To provide a valid comparison between coherentand spontaneous Raman scattering, we employ athree-color CSRS approach similar to that of Petrovet al. [8], depicted in Fig. 1. We use CSRS rather thanCARS to allow for a straightforward experimentalcomparison with the spontaneous Stokes scatteringsignal. A time delay is placed between the pumppulses, which act to generate Raman coherences inthe medium, and the probe pulse, which scattersfrom the Raman coherences. The time delay sup-presses the NR background present during pump–probe overlap. By blocking the pump beams, the ex-periment can be easily switched between coherentand spontaneous methods while maintaining experi-mental variables such as the detector alignment andfocusing conditions.

The laser source was a Ti: sapphire oscillator (Fem-tolasers), with a repetition rate of 75 MHz, pulse en-ergy of 12 nJ, and 110 nm of bandwidth, centered at780 nm. The laser was sent into a 4f pulse shaper

Fig. 1. (Color online) (a) Energy level diagram for time-delayed CSRS. (b) Spectrum used for the CSRS experi-ment. The two pump beams are blocked for the SpRS mea-

surements. (c) Experimental setup.

2009 Optical Society of America

774 OPTICS LETTERS / Vol. 34, No. 6 / March 15, 2009

where a mask selected the spectral components forthe measurements (see Fig. 1). The pump spectrawere chosen to excite the 1000 cm−1 mode of polysty-rene. The spectral resolution of both measurementsis determined by the probe bandwidth �40 cm−1�. Aglass slide delayed the probe pulse by 800 fs to sup-press the NR background, resulting in �20% reso-nant signal loss in the CSRS experiment. For theSpRS measurements, the pump pulses were blocked.The power was measured at the sample, and a vari-able neutral density filter was used to obtain equiva-lent total power for both measurements. The pumpand probe pulses were focused by an air objective(NA 0.4, Olympus LM plan IR, 20�). The pulses werecharacterized by second-order autocorrelation, indi-cating pulse durations within 10% of the transformlimit of 124 fs for pump 1, 132 fs for pump 2, and668 fs for the probe pulse. A scanning stage movedthe sample to obtain 2D images. The signals were col-lected by optics with an NA of 0.6 and directed into aspectrometer (Jobin Yvon IHR320) where spectrawere acquired with a Pixis 100F camera.

Figure 2 shows CSRS and SpRS images of polysty-rene beads dried onto a coverslide, where the differ-ence in spectral intensity at 1000 and at 950 cm−1 de-fines the pixel intensity. Figures 2(a) and 2(b) aredirect comparisons of 4.3 �m bead images usingCSRS and SpRS respectively with 1.3 mW inputpower. In the CSRS and SpRS images the definitionof the bead edges is different owing to the respectivequadratic and linear dependence of the signal onsample length. The averaged spectra shown in Fig.2(f) were measured at the center of the beads, show-ing similar peak intensities and signal-to-noise ratiosfor the CSRS and SpRS measurements. Therefore wedetermine the critical power above which coherentRaman provides larger signal than incoherent meth-ods to be �1.3 mW for solid polystyrene. With an op-timized power ratio of the three pulses (1:1:1) for theCSRS measurement, the critical power can be re-duced to �0.7 mW. We also performed power-dependent measurements in 2-propanol solution un-

Fig. 2. (Color online) Comparison of coherent and SpRSimaging of polystyrene beads. (a) CSRS image of 4.3 �mbeads and (b) corresponding SpRS image using 1.3 mW to-tal power, 200 ms/pixel for both measurements. (c) CSRSimage of the same sample (4 mW total power, 40 ms/pixel).(d) CSRS image of 7.3 �m beads and (e) correspondingSpRS image (1.3 mW total power, 100 ms/pixel). (f)Averaged CSRS (solid) and SpRS spectra (dashed) from 400

pixels at the bead centers in images (a) and (b).

der the same focusing conditions, verifying therespective linear/cubic dependence of the SpRS/CSRSsignals on excitation power.

In a review of CARS, Tolles et al. compare the sig-nal strengths of coherent and spontaneous Ramanmeasurements [6]. Employing Eqs. (4), (5), (19), and(27) from their paper, we find the following expres-sions to describe the power of SpRS and CSRSsignals:

PRaman = � c

8���Epr�2ALN

d�

d��coll, �1�

PCSRS = �0

� �2��CSRS2

cns2 �L2A�3

8�0

�

�3�B��

�Epr�� + �d�2

d�, �2�

where, in cgs units, Epr is the probe electric field, A isthe cross-sectional area of the excited region, L is thesample interaction length, N is the number density ofmolecules, d� /d� is the differential Raman scatter-ing cross section, and �coll is the solid angle collectedin the SpRS measurement. In Eq. (2), �CSRS is thefrequency of the emitted Stokes radiation, nS is theindex of refraction at the Stokes wavelength, �3�

=�v� / 3��v2−2− i��, where = ��1−�2�, �v is the

resonant vibrational frequency, � is a damping con-stant related to the FWHM of the linewidth suchthat �=2� /31/2, and �=2Nc4d� /d� / ���CSRS

4 �. Theterm B��=�0

�E1���+�E2*����d��, where E1 and E2

are the electric fields of pumps 1 and 2. Equation (2)assumes perfect phase matching for the CSRS pro-cess. Based on Eqs. (1) and (2), we estimate the ratioof coherent to spontaneous Raman signals under ourexperimental conditions. The coherent measure-ments used pump power of P1=0.93 mW, P2=0.29 mW, probe power Ppr=0.08 mW, while theSpRS measurement used equivalent total power�Ppr=1.3 mW�. We note that the CSRS signal willcontain SpRS contributions from the probe, esti-mated to be �6% of the total CSRS signal. Otherparameters were focal diameter =1.4±0.1 �m, confo-cal parameter=8.0±0.4 �m, polystyrene density=1.05 g/cm3, L=4.3±0.1 �m, nS=1.55, and �coll=1.45±0.4 sr. Here we have not applied the solidangle correction of 1/ns

2 [12], since it is not appropri-ate for our spherical sample geometry. Using Fouriertransform CARS measurements [5] and comparingsignal levels between polystyrene and pyridine [13],we measured the differential Raman cross section ofthe 1000 cm−1 band of polystyrene to be d� /d�=2.4�10−30±0.6�10−30�cm2/sr� /molecule and itslinewidth to be =8±2 cm−1. This value is for excita-tion at 836 nm and accounts for the difference inscattered wavelength of the pyridine measurement�513 nm� and our observations �913 nm� [13]. Usingthe above numbers we obtain PCSRS/PRaman=6±3,

which is reasonably consistent with our measure-

March 15, 2009 / Vol. 34, No. 6 / OPTICS LETTERS 775

ments and with recent reports comparing CSRS andSpRS [8,9].

We note that the polystyrene measurements weremade on pure samples with �10 M concentration. Inbiological imaging, local concentrations will be sig-nificantly lower, likely within or below the millimolarrange. This will raise the critical power necessary forenhancement with CSRS, owing to the quadratic de-pendence of CSRS on N as opposed to the linear de-pendence of SpRS. For biological applications, the ad-vantage obtainable with coherent methods willdepend critically on the sample’s concentration anddamage threshold. Figure 3 explores the dependenceof spontaneous and coherent signals on concentrationand power. The solid line indicates where the signalsare equivalent, based on our polystyrene measure-ments and assuming that PCSRS/PRaman�NP2. Figure3 does not apply to heterodyne-detected or stimulatedRaman scattering signals, which scale differentlywith N and P. The dashed line shows a projectedequivalence line, adjusted for NA 1.2 and the opti-mized power ratio for CSRS. This adjustment as-sumes an interaction length L of half the confocal pa-rameter for both measurements and will bias theestimate against SpRS, which has a larger effectiveinteraction length. Also indicated are limiting powersat which photodamage in cells becomes significant.The area below both lines is where spontaneousRaman signal levels are higher, while in the grayshaded region above the lines, coherent methods pro-vide larger signal levels. Much of the region wherecoherent methods have a significant advantage is in-accessible in biological samples owing to photodam-age or the requirements of high concentration. Based

Fig. 3. Dependence of the ratio of CSRS/SpRS signal as afunction of power and concentration. The solid line indi-cates equal coherent and spontaneous signals, correspond-ing to the measurement on 4.3 �m polystyrene beads (star)and the expected power and concentration dependence. Theprojected equivalence line for NA 1.2 and an optimizedpower ratio for CSRS is also shown (dashed). Arrows indi-cate power measurements determined to induce cellularphotodamage: a, 2–3 mW, 150 fs pulses, 80 �s dwelltime[11]; b, 4.5 mW, 240 fs pulses duration, 60 �s dwelltime; c, 7.3 mW, 2.2 ps pulse duration, 60 �s dwell time[10]. The dotted box indicates the area most relevant forbiological imaging.

on our calculations, we do not expect Fig. 3 to changesignificantly with picosecond excitation. At low con-centrations and low power, spontaneous methods of-fer a sensitivity advantage. SpRS also has the advan-tage of providing high-resolution broadband spectra,free from NR background. A disadvantage of sponta-neous methods is that they cannot take advantage ofheterodyne schemes that have been shown to providemodest signal-to-noise improvements [14]. Other dis-advantages are the reduction in spatial resolutionand 3D sectioning ability, and possible spectral over-lap with fluorescence. A confocal geometry couldlargely address these latter concerns.

In summary, we have compared imaging based oncoherent and SpRS. In contrast to previous studies,here we use conditions relevant for biological imag-ing. The critical power that generates equal amountsof spontaneous and coherent signal is determined tobe in the milliwatt range for pure samples and will beconsiderably higher at lower concentrations. Thisstudy provides a basis for evaluating the relative per-formance of coherent and spontaneous Raman meth-ods in biological imaging. Depending on the desiredspatial and spectral resolution, and the nature of thesample, the benefits of each method should be criti-cally examined to make an optimum choice of tech-nique.

The authors gratefully acknowledge funding fromthe National Science Foundation (NSF) (grant0721370), the Alfred P. Sloan Foundation.

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