858 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 11, NO. 4, JULY/AUGUST 2005 Optical Trapping and Coherent Anti-Stokes Raman Scattering (CARS) of Submicron-Size Particles James W. Chan, Heiko Winhold, Stephen M. Lane, and Thomas Huser

Abstract—Optical trapping combined with coherent anti-Stokes (∼ 10−30 cm2) that limit its practical use for imaging applica- Raman scattering (CARS) spectroscopy is demonstrated for the tions. The orders of magnitude higher signals in CARS mi- first time as a new technique for the chemical analysis of individ- croscopy allow for rapid CARS imaging approaching video rate ual particles over an extended period of time with high temporal resolution. Single submicron-size particles suspended in aqueous speed over long time periods, which enables monitoring of rapid media are optically trapped and immobilized using two tightly dynamic processes [5], [6] that are otherwise not feasible with focused collinear beams from two pulsed Ti:Sapphire laser conventional fluorescence or Raman microscopy. sources. The particles can remain stably trapped at the focus for In the CARS technique, two laser beams at two different many tens of minutes. The same generate a CARS vibra- frequencies (ω , the pump frequency and ω , the Stokes fre- tional signal from the molecular bonds in the trapped particle when p s the laser frequencies are tuned to a vibrational mode of interest, quency) are tightly focused into the sample to generate a co- providing chemical information about the sample. The technique herent anti-Stokes signal at frequency 2ωp − ωs . Strong reso- is characterized using single polystyrene beads and unilamellar nant CARS signals are generated when the frequency differ- phospholipid vesicles as test samples and can be extended to the ence ωp − ωs matches a Raman-active molecular vibration in study of living biological samples. This novel method could poten- the sample, which induces collective oscillations of the molec- tially be used to monitor rapid dynamics of biological processes in single particles on short time scales that cannot be achieved by ular bonds. Because CARS is a nonlinear multiphoton pro- using other vibrational spectroscopy techniques. cess, the signal is only generated within the focal volume, providing high spatial resolution akin to other multiphoton Index Terms—, coherent anti-Stokes Raman scat- tering (CARS), laser tweezers, . techniques, such as two- fluorescence and second har- monic generation. A CARS image is generated by rapid scan- ning of the laser focus over the entire biological sample. An I. INTRODUCTION additional benefit of CARS microscopy is that sample aut- OHERENT anti-Stokes Raman scattering (CARS) mi- ofluorescence does not usually interfere with the CARS sig- C croscopy has been developed recently [1]–[4] as a tech- nal, which is located at shorter than the excitation nique capable of high spatial resolution for the chemical imag- wavelengths. ing of live biological samples without the need for intrusive The long-term CARS analysis of submicron-size biological labeling with fluorescent probes. Contrast in CARS imaging particles suspended in aqueous media is difficult, if not impos- is generated from vibrational signals of Raman active molec- sible, due to the constant of the particle in ular vibrations in the sample itself. Consequently, a major and out of the laser focus. Optical tweezers [7]–[11] have been benefit of CARS imaging is that the signal, unlike the sig- shown to be able to immobilize a particle in solution such that nal from fluorescent probes, does not photobleach, enabling it remains within the focal volume of the focused laser beam, long-term imaging of a living sample as it undergoes biolog- essentially “trapping” the particle indefinitely. This trapping is ical processes. The coherent nature of the nonlinear CARS enabled by from the laser beam imparting their mo- process results in vibrational signals that are much stronger mentum to the particle, which results in transverse and axial than spontaneous Raman signals. Spontaneous Raman spec- forces that are balanced near the laser focus. Several more re- troscopy is known for its relatively low scattering cross-sections cent studies [12]–[18] have already demonstrated the ability to combine optical trapping with spontaneous Raman spectroscopy using (CW) laser sources, which allows for the Manuscript received December 17, 2004; revised June 15, 2005. This work chemical analysis of biological specimens and their dynamics. was supported by the National Science Foundation (NSF). The Center for Bio- However, this laser tweezers Raman spectroscopy (LTRS) tech- photonics, an NSF Science and Technology Center, is managed by the University of California, Davis, under Cooperative Agreement No. PHY 0120999. Work at nique still suffers from the inability to acquire spectra rapidly LLNL was performed under the auspices of the U.S. Department of Energy by enough to monitor fast dynamics. Typically, spectra have to the University of California, Lawrence Livermore National Laboratory, under be acquired over periods of many seconds to minutes, thus al- Contract No. W-7405-Eng-48. The authors are with Lawrence Livermore National Laboratory, Livermore, lowing only slow, long-term dynamics (e.g., apoptosis) to be CA 94550 USA, and also with the NSF Center for Biophotonics Science studied. CARS signals, in contrast, being that much stronger and Technology, University of California-Davis, Sacramento, CA 95817 USA than spontaneous Raman signals, would enable monitoring of (e-mail: [email protected]; [email protected]; [email protected]; huser1@ llnl.gov). fast dynamics with higher temporal resolution and maintaining Digital Object Identifier 10.1109/JSTQE.2005.857381 of chemical specificity.

1077-260X/$20.00 © 2005 IEEE CHAN et al.: OPTICAL TRAPPING AND (CARS) SPECTROSCOPY OF SUBMICRON-SIZE PARTICLES 859

spectrometer (Ocean , Dunedin, FL) with a resolution of roughly 1 nm. The parallel-polarized beams are expanded with a telescope assembly to approximately 5 mm and sent into an inverted optical microscope (Axiovert 200, Zeiss) equipped with a dichroic mirror and a 100 ×, 1.3 NA oil immersion objective (Plan-NEOFLUAR, Zeiss, Germany). CARS signals generated at the laser focus are epi-detected and sent through a 100-µm confocal pinhole and a short pass filter for rejection of the excitation light. The CARS signal is detected with either an avalanche or a grating spectrometer equipped with a liquid nitrogen cooled 1340 × 100 pixel charge-coupled device (CCD) camera (Roper Scientific, Trenton, NJ). The system is also equipped with a separate 30-mW He-Ne laser at 633 nm that is capable of trapping single particles and functions as an excitation laser for acquiring spontaneous Raman spectra. In addition, a pulse picker (NEOS Technologies, Melbourne, Fig. 1. Schematic of the Raman and CARS confocal detection system. Two FL) can be added right after both beams are combined by the pulsed Ti:Sapphire laser beams are collinearly combined into an inverted mi- croscope. Detection of the spectral signals is achieved using a standard confocal dichroic reflector to reduce the repetition rate of each laser geometry, an avalanche photodiode, and spectrometer. A CW He-Ne laser is beam down to 200 kHz. This reduces the average power of also implemented for spontaneous Raman measurements, and a pulse picker is the beams and minimizes photodamage to samples, while used to reduce the repetition rate of the Ti:Sapphire beams. maintaining high peak powers for CARS signal generation.

Optical trapping with pulsed laser systems has not been as B. Preparation of Unilamellar Vesicles widely demonstrated as with CW lasers but has been gaining 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) is considerable attention due to the ability to study a trapped purchased in powder form from Avanti Polar Lipids, Alabaster, particle by nonlinear spectroscopic methods that require intense AL. Liposomes are prepared using standard techniques [23]. short laser pulses. Modeling studies [19] have predicted the Briefly, a DMPC/chloroform solution is dried under nitrogen for feasibility of ultrashort-pulsed laser tweezers, and experimental several minutes, placed under overnight to remove trace studies have demonstrated the combination of trapping with quantities of chloroform, and hydrated above its lipid transition SHG [20], [21] and two-photon fluorescence [22]. In this temperature. Unilamellar vesicles are prepared through extru- paper, we demonstrate the combination of optical trapping with sion of the multilamellar vesicle solution using a mini-extruder CARS spectroscopy using two pulsed laser sources at different (Avanti Polar Lipids). Approximately 1 mL of the hydrated lipid wavelengths that are overlapped both spatially and temporally. suspension is extruded several times at its phase transition tem- This technique makes it possible to rapidly obtain chemical perature using polycarbonate membranes (Avanti Polar Lipids) information from submicron-size particles in their natural with a 0.4-µm pore size to yield a concentrated solution of environment. The trapping parameters and CARS signal are 0.4-µm diameter unilamellar vesicles. This solution is then di- demonstrated and fully characterized for polystyrene bead and luted several times to a point where single vesicles can be ob- unilamellar vesicle samples. served and trapped at the laser focus.

II. EXPERIMENTAL METHODS III. TRAPPING OF POLYSTYRENE BEADS A. Optical Raman and CARS Tweezers Setup A drop of solution containing 1-µm polystyrene beads (Duke A schematic of the setup is shown in Fig. 1. Two 80-MHz Scientific Corp., Palo Alto, CA) is placed on a coverslip, which 5-ps pulsed tunable Ti:Sapphire lasers (Tsunami, Spectra- rests on a piezoelectric translation stage [Fig. 2(a)]. A single Physics, Mountain View, CA) are temporally synchronized by bead is stably trapped at the tight focus of the two collinearly phase-locked loop electronics (Lok-to-Clock, SpectraPhysics) combined laser beams, as shown in the white light image in and collinearly combined with a dichroic mirror. Both lasers Fig. 2(b), whereas other particles in the field of view are ob- are tunable from 750 nm (13333.33 cm−1) to 900 nm (11111.11 served in constant motion and out of focus. It is known [2] that cm−1). The entire Raman spectral window from 500 to 2000 the intensity of epi-detected CARS signals is strongest for small cm−1 can be covered, which is the molecular fingerprint region scatterers when the diameter is on the order of the of interest for biological samples where most information about of the excitation light. Larger beads up to 4 µm can also be cellular constituents (e.g., DNA, , lipids) can be found. stably trapped. Typically, a single particle that is roughly 5 µm Clean-up filters are used to suppress the amplified spontaneous away from the laser focus will be drawn toward the laser focus emission from the Ti:Sapphire lasers within the spectral range due to the intensity gradient of the laser beam and become opti- where it can interfere with detection of the CARS signals. Laser cally trapped. The particle is stably immobilized approximately power is controlled with polarization optics. The laser wave- 10 µm above the coverslip surface. Attempts to trap particles that lengths are measured using a broadband fiber-optic handheld are further away from the glass surface proves difficult, possibly 860 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 11, NO. 4, JULY/AUGUST 2005

Fig. 2. (a) Schematic of optical trapping at the laser focus. (b) White light image of a single trapped 1-µm polystyrene bead (circled) by two spatially and temporally overlapped pulsed laser beams. Other particles are observed out of focus in the background. Scale bar is 5 µm. due to the distortion of the laser focus as it probes deeper into water. High laser powers (50 mW total) result in trapping for tens of seconds, at which point the particle will shoot out of the trap axially. It is not clear at this point whether this phenomenon is due to the instability of the trap at such high powers or laser damage to the bead, which results in potential bead deforma- tion and increased absorption due to chemical modifications. When the power is reduced to 30 mW and below, the trap is Fig. 3. (a) CARS signal at 697 nm and 703 nm when the lasers are tuned, much more stable and the bead remains immobilized for many on resonance to a molecular vibration at 1001.5 cm−1 and off resonance at −1 minutes. Once trapped, the particle can be manipulated in the 900 cm for a single trapped polystyrene bead. The graph shows the reso- nant enhancement of the CARS signal over the nonresonant background signal. solution transverse to the beam by moving the translation stage (b) Log plots showing the squared and linear dependence of the CARS signal on and a few microns axially by moving the objective. the pump and Stokes laser intensity, further confirming that CARS is detected. (c) Plot of the CARS signal intensity as a function of time for a single trapped bead. The plot demonstrates the ability to obtain signals on the order of millisec- IV. CARS CHARACTERIZATION OF TRAPPED BEADS onds and the overall stability of the signal on the second time scale. Fluctuations on the millisecond time scale are attributed to jitter of the phase-locked loop Fig. 3(a) shows the intensity of the CARS signal when the two electronics of the laser system. lasers are tuned on resonance to the 1001.5 cm−1 vibration of the polystyrene bead (ωp = 750 nm, ωs = 811 nm), which gives factor of roughly five times the nonresonant background signal. a CARS signal positioned at 697 nm. Typical pump and Stokes No signal was obtained in the absence of a particle within the laser powers are 20 and 10 mW before the microscope objective trap. The signals are confirmed to be CARS signals by deter- and the spectra are acquired in 50 ms. No signal deterioration mining the dependence of the signal on the pump and Stokes is observed, indicating that no bead modification or damage is laser intensity, which is known to have a squared and linear ∼ 2 occurring at these powers. In addition, the bead damage occur- dependence, respectively (ICARS IpIs ) [1]. Fig. 3(b) shows ring at higher laser power is usually accompanied by an intense, the log plots of the CARS intensity versus the pump and Stokes broad fluorescence. We do not observe this fluorescence dur- laser power and the slope values, which confirm the squared and ing the entire time the bead is maintained in the laser focus. linear dependence. For comparison, when the lasers are tuned off any polystyrene To confirm the stability of the trap and the signal from the resonance vibration (e.g., at 900 cm−1), a weaker nonresonant particle, which is important if dynamic biological processes are background signal that is inherent in CARS spectroscopy [4] is to be inferred from small signal changes in the CARS signal detected at 703 nm. The on-resonance signal is stronger by a intensity, the signal was acquired for many tens of seconds. CHAN et al.: OPTICAL TRAPPING AND (CARS) SPECTROSCOPY OF SUBMICRON-SIZE PARTICLES 861

Fig. 3(c) shows the CARS signal from a trapped bead acquired cation, particles are allowed to diffuse naturally into the focal for 10 s every 50 ms. The signal average is 600 counts with volume of the CARS excitation beams, whereupon a CARS a standard deviation of 18 counts. On the seconds time scale, signal burst will be detected. Detection of these signal bursts the signal is stable; however, signal fluctuations are observed in and an autocorrelation analysis provides information about the the millisecond regime. We attribute these fluctuations mainly diffusion dynamics of the particles. It should be noted that it is to the timing jitter between the two laser beams that is inherent important that the laser beams not influence the natural diffu- in the system. These fluctuations can be eliminated by using sion of the beads in order for accurate dynamics to be recorded. more sophisticated timing electronics to reduce this jitter, as has In their study, laser beams with a repetition rate of 400 kHz been demonstrated by Potma et al. [24] or by using other laser were used at average powers slightly lower than the powers we systems that do not rely on electronic locking of two independent reported above for trapping of particles. Their study also used laser systems, such as an optically parametric oscillator (OPO) smaller particles as well (175-nm particles). There is clearly a system. It is possible that the fluctuations are also a result of power level above which the laser forces influence the particle variations of the bead position within the trap. motion and below which there is no noticeable effect. Further studies are currently under way to investigate this dependence on laser power and particle size, shape, and optical properties, V. CARS TRAPPING WITH PULSE PICKED BEAMS all of which are important for the CARS-CS technique. For CARS analysis of living biological samples, the use of high repetition rate and high average power pulsed lasers will VI. CARS TRAPPING OF UNILAMELLAR VESICLES result in damage to the sample. It has been demonstrated [2], [4] Previous studies [23], [26], [27] have used laser trapping previously that incorporating pulse picking to reduce the average with Raman spectroscopy to obtain the Raman vibrational fin- power of the laser beam while maintaining high peak pulse in- gerprint of unilamellar vesicles. CARS spectroscopy has also tensity, which is the important parameter for generating a CARS been applied to the study of lipid bilayers and liposomes on a signal, can circumvent the issues of photodamaging biological substrate [28]–[31] Here, we demonstrate the optical trapping samples. Here, we investigate the feasibility of maintaining sta- of unilamellar vesicles and the acquisition of CARS signals at ble optical trapping using lower powers with pulse picked laser a specific molecular vibration. beams and the simultaneous acquisition of CARS signals. A Single vesicles can be trapped stably (at 80 MHz) using as pulse picker is used to reduce the repetition rate of the laser beam little as 10 mW total laser power. Because the vesicles are only to 4 MHz, 2 MHz, 800 kHz, and 400 kHz. At these repetition 0.4 µm in diameter, often several vesicles will end up clustering rates, the peak pulse energy can be maximized for CARS exci- into the laser focus. Typically, around five vesicles are observed tation (∼5 nJ as opposed to ∼0.5 nJ for the data in the previous to be clustering at one time, which can be avoided by diluting section), while maintaining low average powers of 20, 10, 4, and the vesicle solution appropriately. Fig. 4(a) shows the Raman 2 mW, respectively. These powers are also sufficient for stable spectrum from a single unilamellar DMPC vesicle trapped and trapping and simultaneous CARS acquisition and are consistent excited using the 633-nm CW laser. The peaks are consistent with previous studies [20] showing that pulsed repetition rate with previously reported Raman spectra of DMPC vesicles. The lasers as low as 25 kHz can still optically trap nanometer-size 1440 cm−1 peak, assigned to a CH bending mode of the lipids, particles as long as the frequency of the laser pulses surpasses 2 is chosen to generate the CARS signal and is wide enough to the time it takes for the particle to move beyond its root mean enable tuning of the lasers over the entire width of the peak square (rms) displacement from its equilibrium position. [full-width at half-maximum (FWHM) 40 cm−1)] to trace out To determine whether the CARS signal fluctuations at the the profile of a CARS spectrum. Because we are currently lim- millisecond time scale, such as those observed in Fig. 3(c), ited by the resolution of the broadband spectrometer used to could be attributed to bead motion within the optical trap, we measure the laser wavelength, we are only able to collect five need to approximate the rms value of the particle position. It data points. Each data point is an average of many CARS signals has been reported that 80-nm particles have an rms value of 20 collected from different trapped unilamellar vesicles. The laser nm for 40 µs, which corresponds to 25 kHz [20]. For the 1-µm power of the pump (750 nm) and Stokes (841 nm) laser beams particles and the 400 kHz–80 MHz laser repetition rates that are are 30 mW and 15 mW, respectively. Fig. 4(b) shows the CARS used in this study, the rms values would be even smaller (∼1 spectral response (square points) overlaid on a magnified trace nm and less). Therefore, we determine that the impact of the of the spontaneous Raman peak from Fig. 4(a), which mirrors displacement of the 1-µm bead should not have a significant the Raman vibrational profile of the 1440 cm−1 peak. effect on the signal fluctuations. It should be pointed out that at slightly lower powers than those specified previously, the intensity gradient of the laser VII. CONCLUSION beam still affects the natural Brownian motion of the particle, In conclusion, we have, for the first time, demonstrated drawing it into the focus even though it does not remain sta- the combination of optical tweezers with CARS spectroscopy. bly trapped. This information is of particular interest for appli- CARS signals from individual submicron-size objects such as cation of CARS toward correlation spectroscopy (CS). Cheng polystyrene beads and unilamellar lipid vesicles were obtained et al. [25] reported the use of CARS-CS to probe diffusion with millisecond temporal resolution. CARS tweezers spec- dynamics of nanometer-size polystyrene beads. For this appli- troscopy is a novel method that can potentially be used for 862 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 11, NO. 4, JULY/AUGUST 2005

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[26] D. P. Cherney, T. E. Bridges, and J. M. Harris, “Optical trapping of He is currently a guest at Lawrence Livermore National Laboratory (LLNL), unilamellar phospholipid vesicles: Investigation of the effect of optical Livermore, CA, where he is writing his Master’s thesis. At LLNL, he applies forces on the lipid membrane shape by confocal-Raman microscopy,” laser tweezers Raman spectroscopy and coherent anti-Stokes Raman scattering Anal. Chem., vol. 76, pp. 4920–4928, 2004. spectroscopy to biological and medical problems. [27] J. M. Sanderson and A. D. Ward, “Analysis of liposomal membrane com- position using Raman tweezers,” Chem. Commun., vol. 9, pp. 1120–1121, 2004. [28] J. X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “Multiplex coher- ent anti-stokes Raman scattering microspectroscopy and study of lipid Stephen M. Lane received the Ph.D. degree from the University of California- vesicles,” J. Phys. Chem. B, vol. 106, pp. 8493–8498, 2002. Davis, Sacramento, CA, in 1978. [29] G. W. H. Wurpel, J. M. Schins, and M. Muller, “Chemical specificity in He is currently the Chief Scientific Officer for the National Science Foun- three-dimensional imaging with multiplex coherent anti-Stokes Raman dation Center for Biophotonics at University of California-Davis, where he is scattering microscopy,” Opt. Lett., vol. 27, pp. 1093–1095, 2002. also an Adjunct Professor in the Department of Radiology. In addition, he is [30] E. O. Potma and X. S. Xie, “Detection of single lipid bilayers with coherent the Deputy Division Leader for the Medical Physics and Division anti-Stokes Raman scattering (CARS) microscopy,” J. Raman Spectr., of Lawrence Livermore National Laboratory, Livermore, CA. He has more than vol. 34, pp. 642–650, 2003. 60 publications and 11 patents. He has worked in the areas of nuclear physics, [31] G. W. H. Wurpel, J. M. Schins, and M. Muller, “Direct measurement radiation detection, laser fusion, optical sensors, and photon transport modeling. of chain order in single phospholipid mono- and bilayers with multiplex For the past 14 years, his focus has been on medical device development and CARS,” J. Phys. Chem. B, vol. 108, pp. 3400–3403, 2004. biophotonics.

James W. Chan received the Ph.D. degree from the University of California- Davis, Sacramento, CA, where his work involved using vibrational and fluores- cence spectroscopy to study laser modification in optical material. Thomas Huser received the Ph.D. degree in physics from the University of He is currently a postdoctoral research staff member at Lawrence Liver- Basel, Switzerland, where he worked primarily on near-field optical microscopy. more National Laboratory, Livermore, CA. His current work involves using He currently is a Group Leader for Biophotonics and Nanospectroscopy techniques such as Raman and coherent anti-Stokes Raman scattering (CARS) at Lawrence Livermore National Laboratory (LLNL), Livermore, CA. He is spectroscopy and microscopy for biomedical applications, such as cancer de- also a Theme Area Leader for Bioimaging at the University of California- tection and studying biological processes at the single level. Davis, Sacramento, CA, National Science Foundation Center for Biophotonics Science and Technology. He joined LLNL in 1998 as a Postdoctoral Associate and became a Staff Scientist in 2000. At LLNL, he applies single- Heiko Winhold is pursuing the Master’s degree in medizinisch physikalische fluorescence and single-cell Raman spectroscopy to biological and medical technik/medical engineering at the University of Applied Sciences Berlin (TFH). problems. He has published more than 30 papers in peer-reviewed journals.