trends in analytical chemistry, vol. 20, no.

5, 2001

255

Near-infrared Raman spectroscopy of single particles

Katsuhiro Ajito*, Keiichi Torimitsu
NTT Basic Research Laboratories, Nippon Telegraph and Telephone Corporation, 3-1, Morinosato-Wakamiya, Atsugi, Kanagawa 243-0198, Japan
Raman spectroscopy using non-invasive near-infrared (NIR ) laser light has become a powerful tool for the microscopic analysis of organic and biological materials. A Raman tweezers microscope (RTM ) was developed by combining NIR Raman spectroscopy with the laser trapping technique, which enables us to expand the scope of single particle studies. Recent results obtained using the RTM for single droplets and polymer spheres in a micrometer range are reported. z2001 Elsevier Science B.V. All rights reserved.
Keywords: Raman microscopy; Laser trapping; Optical tweezers; Near-infrared; Microparticle; Microdroplet; Picoliter; Liquid^liquid extraction

1. Introduction
The study of single particles is attractive from the viewpoint of identifying chemical compositions, such as species, structures, and conformations of molecules in individual particles, and revealing molecular behavior in such very small spaces. The chemical reactions in particles are different from those in bulk, because the ratio of the surface area to the volume of a particle is much larger than that of a conventional container for bulk materials. Single particle studies have progressed with the development of techniques that allow us to manipulate single particles, such as the electrodynamic balance ( EDB ) technique and laser trapping. The EDB technique based on ac and dc current electric elds can be used to trap one or more charged aerosol particles [ 1 ]. Raman spectroscopy and infrared absorption spectroscopy have been used to study inor-

*Corresponding author. Fax: +81 (462) 70-2364. E-mail: ajito@will.brl.ntt.co.jp

0165-9936/01/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 0 1 ) 0 0 0 6 0 - 7

ganic ions in EDB-trapped aerosol particles. Absorption spectroscopy was applied to obtain the molecular composition of a single trapped particle; however, spectral resolution was not sufcient for quantitative analysis of the particle [ 2 ]. Raman spectroscopy provides high spectral resolution and enables us to determine concentrations of environmentally important inorganic ions contained in aerosol particles such as nitrate and sulfate ions in single droplets in gases [ 3,4 ]. Several approaches have been utilized to enhance the Raman scattering from aerosol particles. Morphology-dependent resonances (MDRs ) have been used to elastically enhance Raman scattered light whenever the size and refractive index of the particle lead to interference of the light waves in the particle [ 5 ]. For the MDR technique, broad bands like the OH- or CH-stretching vibrational bands are necessary. Furthermore, a resonance Raman scattering technique has been shown to extremely enhance the Raman scattered light from dyes in aerosol particles [ 6 ]. The laser trapping technique has been widely used to capture and manipulate single organic particles in a micrometer range, such as latex beads, aerosol particles, microdroplets, microcapsules, and biological samples ( bacteria, blood cells, etc. ) [ 7^10 ]. Laser trapping, by which a small particle is grabbed by the force of radiation pressure generated from two laser beams, was rst reported in 1970 [ 11 ]. Then a more practical method using one laser beam with a microscope was introduced, which is called the optical tweezers [ 12 ]. The optical tweezers initially used a visible laser beam. Nowadays though a low-energy near-infrared (NIR ) laser beam ranging from 700 to 1100-nm wavelength is widely used because it produces far fewer sample-damaging photochemical reactions than visible laser light. An NIR laser beam focused on a small particle using an objective lens traps the particle without damaging it. Another advantage of this technique, one that is particularly attractive to
2001 Elsevier Science B.V. All rights reserved.

256

trends in analytical chemistry, vol. 20, no. 5, 2001

biological scientists, is that it allows the manipulation of not only cells but also of organelles and vesicles within cells [ 13,14 ]. The optical tweezers have been used with spectroscopic techniques to characterize molecules contained in single particles. Chemical reactions within single microdroplets or microcapsules have been studied using systems that combine laser trapping with electrochemistry [ 15 ], uorescence spectroscopy [ 15,16 ], absorption spectroscopy [ 16 ], or Raman spectroscopy [ 17^19 ]. Raman spectroscopy is advantageous as an analytical tool for trapped particles because it provides information about species, structures, and conformations of various kinds of molecules in the particle. Another advantage regarding instrumentation for Raman measurements is that the focused laser beam can be used for both laser trapping and Raman spectroscopy. Visible laser light has been widely used as an excitation light source for Raman spectroscopy to study single organic particles [ 17^21 ]; however, non-invasive NIR laser light has not been widely used until very recently. This is because Fourier transform Raman spectroscopy [ 22^25 ], a typical Raman spectroscopic technique using NIR laser light, is difcult with conventional optical microscopes and has a much lower sensitivity than visible Raman spectroscopy. Nevertheless, a highly sensitive Raman spectroscope using NIR laser light is required in microchemistry, mesoscopic chemistry, and biological engineering for analyses of trapped particles, because the NIR laser light eliminates uorescent background in the Raman spectra of organic and biological materials. Recently, we developed a new NIR Raman microscope comprising a high-power NIR laser, an optical microscope, holographic notch lters ( HNFs ), a single-grating polychromator, and a charge-coupled device ( CCD ) camera. Then, we demonstrated a system that combined NIR Raman microscopy with the laser trapping technique, which is called the Raman tweezers microscope (RTM ) [ 26 ]. This paper describes the application of the RTM for the analysis of single particles in the micrometer range. The advantage of the RTM is that it can trap single particles without damaging them and provides a wealth of molecular information about those particles. The paper presents the recent results of a RTM analysis of tiny particles and also discusses the difference in behavior between a molecule in a single droplet and one in bulk.

2. Apparatus and principles

Fig. 1 is a schematic of the RTM system. An NIR laser beam is used for both laser trapping and Raman spectroscopy in the system. The laser trapping technique employs the force of gradient radiation pressure generated when a laser beam is tightly focused onto a small particle under an optical microscope. Although small particles in solution are always moving by Brownian motion, they can be trapped by using this technique. When the laser beam is focused short of the center of the particle, the laser light is refracted at the surface of the particle and its momentum is changed. The change of the momentum causes the force of radiation pressure to pull the particle toward the beam ( shown in the particle model illustrated in Fig. 1 ). Focus the beam beyond the center of the particle and the particle will be pushed away, while a focal point to the left or right of center would cause the particle to move left or right. Consequently, the particle is completely trapped by the optical force. The experimental apparatus is described briey here. The laser light source is a continuous-wave, single-frequency titanium:sapphire laser (TitanCw, Schwartz Electro-Optics ) tuned from 730 to 780 nm in the TEM00 mode. The pump source for the titanium:sapphire laser is the 532-nm line of a solid-state continuous-wave laser (Millenia, Spectra-Physics Lasers ). A commercial Raman microprobe spectrometer (Ramascope, Renishaw ) was specially modied for NIR laser light. The system is controlled by a Windows-type personal computer. The expanded NIR beam is focused onto the sample using an objective lens with 100U magnication and a numerical aperture of 0.8. The lens is mounted on an optical microscope ( BH-2, Olympus ). The objective lens used to focus the laser onto the sample is also used to collect light scattered from the sample at 180 with respect to the incident light. After the scattered light passes through two HNFs to remove Rayleigh scattered light, it is focused onto the entrance slit of a single-grating polychromator, which is then focused onto a CCD camera ( 02-06-1-225, Wright Instruments ). This CCD camera contains a Peltier-cooled slow-scan 384U576 CCD chip maintained at V200 K. In the original system, the CCD camera that records a Raman spectrum was also used to obtain an optical image of a trapped particle [ 26 ]. However, in the new version, the microscope was tted with an additional CCD camera and a HNF [ 27 ]. A

trends in analytical chemistry, vol. 20, no. 5, 2001

257

Fig. 1. Diagram of the RTM system and the optical path of the laser beam in a small particle during laser trapping.

dielectric multilayer-coated beam splitter in the microscope divides the scattered NIR light into two paths, one for each CCD camera. The two-slit confocal arrangement [ 28^30 ] is used to eliminate Raman scattered light from the outer region of the particle. The rst slit is the entrance slit of polychromator and the second slit is the very narrow readout window for the CCD camera. The slits are perpendicular to each other. Using these slits instead of pinholes makes it easier to make the optical alignments needed for confocal Raman measurements.

3. Experiments and results

3.1. Raman spectroscopy of a trapped droplet
The RTM enables us to obtain Raman spectra from a single tiny droplet. Fig. 2 illustrates the optical arrangement for a toluene droplet in a hemi-

spherical water drop xed in the sample cell [ 26 ]. Toluene was selected as the solvent for droplets because it has a much larger refractive index ND (ND =1.497 at 293 K ) than water (ND =1.333 at 293 K ). The higher ND leads to a larger optical radiation force in water. The droplets were made from a toluene^water mixture by an ultrasonic treatment. The toluene droplets gradually aggregated at the center of the water drop at the surface. Therefore, the laser trapping was carried out at the water drop surface away from the area where the droplets gathered. The laser spot focused using the objective lens was V1 Wm in diameter at a power of about 80 mW. A single toluene droplet about 15 Wm in diameter was trapped in the vertical direction almost immediately and was completely trapped in the lateral direction within 20 s after laser illumination was started. The Raman spectrum for the single trapped toluene droplet was clearly obtained below 100 cm31 to above 3000 cm31 as shown in Fig. 2. The

258

trends in analytical chemistry, vol. 20, no. 5, 2001

Fig. 2. NIR Raman spectrum of a single toluene droplet in water and a schematic of the optical arrangement for the laser-trapped droplet in the sample cell.

total exposure time in this scanning range was 5 s. Noteworthy is the absence of uorescence interference in the Raman spectrum due to the use of NIR laser light. Furthermore, the signal-to-noise ratio of the spectrum is sufcient to identify the molecular species of a single droplet. A Raman spectrum obtained by using RTM can be applied to the quantitative analysis of a single trapped particle [ 27 ]. Sample droplets were made from a p-cresol toluene mixture with ultrasonic treatment in deionized water and mainly ranged from 10 to 20 Wm in diameter. The image in Fig. 3 shows the droplets in water under the objective lens. One droplet was trapped by the laser probe ( upper right ) and the other droplet was free and moving by Brownian motion ( lower left ). The bright spot in the trapped particle is the focal spot of the laser beam at a power of 120 mW, visualized using the additional CCD camera. The intensity of the NIR light reected from the focused spot is much higher than the white light reected from the droplet. However, the HNF reduces the inten-

Fig. 3. Image of droplets made from the p-cresol toluene mixture and the focal spot of the NIR laser beam.

trends in analytical chemistry, vol. 20, no. 5, 2001

259

3.2. Molecular extraction in single droplets

Fig. 5 shows our recent results concerning the liquid^liquid extraction process in a single subpicoliter droplet [ 31 ]. Fig. 5A shows the set of the timedependent images of a trapped single subpicoliter droplet in water. The four images are of the same area. The image at the far left shows a single toluene droplet just after it was trapped. The trapped droplet is V10 Wm in diameter, which corresponds to a volume of V0.52 pl when the droplet is spherical. The bright spot in the center of the trapped droplet is the focal point of the laser beam. The other images show the trapped droplet 1, 2, and 3 min after pnonylphenol (PNF ) was added to the solution. Fig. 5B shows the time-dependent Raman spectra of the trapped droplet in the images in Fig. 5A. The exposure time for each spectrum was 3 s. The sharp peaks at 785, 1003, 1030 cm31 are attributed to the modes of the phenyl group of toluene. The four spectra were normalized by using the peaks of toluene at 1003 cm31 . One minute after the PNF was added, the two peaks at 818 and 840 cm31 appeared. These peaks are attributed to the doublet of the ring-breathing mode of PNF. Within 2 min, the PNF peaks increased relative to the normalized peaks of toluene and their intensities saturated after that. These results indicate that the trapped droplet extracted PNF from the solution, which caused the droplet to increase in size as shown in Fig. 5A. The same experiment was done nine more times for droplets of various sizes. Fig. 5C shows the initial droplet size dependence of the concentration and the distribution coefcient of PNF in single subpicoliter and picoliter droplets. The distribution coefcient is dened as the ratio of PNF concentration in the droplet to that in the solution around the droplet. The dashed line indicates the distribution coefcient of PNF in bulk solution. The result indicates the liquid^liquid extraction process in droplets ranging from subpicoliter to picoliter is different from that in bulk solution and the difference is very large for a subpicoliter droplet. This phenomenon can be explained considering the droplet surface areas. The ratio of the surface area to the volume of a subpicoliter droplet is much larger than that of solution in a conventional container, such as a separating funnel, and this strongly affects the surface reactions in the liquid^liquid extraction process.

Fig. 4. NIR Raman spectra of the single trapped droplet ( A ) and bulk solution ( B ).

sity of the scattered NIR light by a factor of about 1034 , which prevents CCD pixel saturation. The Raman spectrum of the trapped droplet is spectrum A in Fig. 4. The exposure time of this Raman spectrum was 3 s. The relative intensity among peaks in a Raman spectrum gives us the concentration ( the mole fraction ) of each species. Spectrum B is the Raman spectrum for the bulk solution, which was made by mixing p-cresol ( 30 mol%) and toluene ( 70 mol%). It was also obtained in the exposure time of 3 s. The peaks at 785, 1003, and 1030 cm31 are attributed to the ring-breathing mode of toluene and the peaks at 823 and 843 cm31 are attributed to the doublet of the ring-breathing mode of p-cresol. These peaks have been observed for a large number of para-substituted benzenes. The spectra were normalized with the toluene peak at 1003 cm31 . The intensities of the peaks for the p-cresol at 823 and 843 cm31 in spectrum A are about ve times smaller than in spectrum B. If the water content in the droplet is neglected, the concentration of p-cresol calculated form the spectrum is V7 mol% in the droplet. This result indicates the RTM system can easily determine the concentration of each molecular species in single particles.

260

trends in analytical chemistry, vol. 20, no. 5, 2001

Fig. 5. Time-dependent images of a single subpicoliter toluene droplet during liquid^liquid extraction of PNF ( A ) and the NIR Raman spectra corresponding to their images ( B ). Initial droplet size dependence of the concentration and the distribution coefcient for the single subpicoliter and picoliter droplets C. The dashed line in C indicates the distribution coefcient of PNF in the bulk solution.

3.3. Raman spectroscopy of smaller single particles

The RTM system can be applied for the analysis of a smaller particle [ 32 ]. The smallest droplet in the experiments described above was V10 Wm because it is difcult to make toluene droplets any smaller than that. The laser probe of the RTM system has about a 1- Wm diameter at the focal plane, which makes it possible to obtain a Raman spectrum from a single particle in the several micrometer range without loss of sensitivity. In this section, the Raman measurement of smaller polymer spheres is shown. Image B in Fig. 6 shows a single polymer sphere trapped in water. The trapped sphere is a polystyrene latex bead about 2.1 Wm in diameter. The refrac-

tive index of the beads is 1.580 ( at 293 K ), which is larger than toluene and makes them easier to trap in water. Image A was recorded in the same area as image B before trapping. Image B shows the single bead trapped in water. There are no other particles in the image because the sample solution was very dilute. The spectra corresponding to these images are also shown in Fig. 6. The exposure time for each spectrum was 5 s. The spectrum corresponding to image A shows the background of the solution and the spectrum corresponding to image B shows the Raman spectrum of the single trapped polymer sphere. The signal-to-noise ratio in the spectrum of the single trapped particle is sufcient to allow identication of the molecular species of the particle. These results indicate the system can trap sin-

trends in analytical chemistry, vol. 20, no. 5, 2001

261

Fig. 6. Images and NIR Raman spectra of a single polystyrene latex bead ( A ) before and ( B ) during laser trapping.

gle particles with diameters as small as several micrometers and provide their Raman spectra. The detection of particles in the submicrometer range, the size range of many kinds of organelles in cells, will be very important in biological engineering. However, it is necessary to increase the sensitivity of the system for such very small particles because the sensitivity decreases with decreasing particle size.

4. Conclusions
The RTM was developed for the characterization of organic and biological single particles. The RTM system, which combines NIR Raman spectroscopy with the laser trapping technique, provides Raman spectra of single trapped particles in a micrometer range and makes it possible to determine the particles' chemical compositions, such as their molecular species and structures. The advantage of the system is that it uses NIR laser light instead of

visible laser light, which prevents photochemical damage to organic and biological particles during laser trapping and results in much lower uorescence interference in the Raman spectra of trapped particles. Our recent experiment showed that the molecular behavior in a single droplet is different from that in bulk solution during the liquid^liquid extraction process, which is due to the restriction of molecular diffusion by the surface of the droplet. With further improvements, the RTM will be able to reveal the features of molecules in single nanoparticles and will open the way to the analysis of biological molecules in very small organelles. We believe that this technique will be widely used in biological engineering in the near future.

Acknowledgements
The authors thank Dr. M. Morita, Dr. H. Takayanagi (NTT Basic Research laboratories ) for their

262

trends in analytical chemistry, vol. 20, no. 5, 2001

encouragement, and Mr. D. Steenken ( Kurdyla and Associates ) for English consultation.

References
[ 1 ] E.J. Davis, Aerosol Sci. Technol. 26 ( 1997 ) 212. [ 2 ] G.S. Grader, S. Arnold, R.C. Flagan, J.H. Seinfeld, J. Chem. Phys. 86 ( 1987 ) 5897. [ 3 ] K.H. Fung, I.N. Tang, J. Colloid Interface Sci. 130 ( 1989 ) 219. [ 4 ] K.H. Fung, D.G. Imre, I.N. Tang, J. Aerosol Sci. 25 ( 1994 ) 479. [ 5 ] J.F. Widmann, C.L. Aardahl, E.J. Davis, Trends Anal. Chem. 17 ( 1998 ) 339. [ 6 ] K.H. Fung, I.N. Tang, Appl. Spectrosc. 46 ( 1992 ) 159. [ 7 ] K. Svoboda, S.M. Block, Annu. Rev. Biophys. Biomol. Struct. 23 ( 1994 ) 247. [ 8 ] M.W. Berns, Sci. Am. 278 ( 1998 ) 62. [ 9 ] M.P. Sheetz, Laser Tweezers in Cell Biology; Methods in Cell Biology, Vol. 55, Academic Press, CA, 1998. [ 10 ] H.-B. Kim, S. Yoshida, A. Miura, N. Kitamura, Anal. Chem. 70 ( 1998 ) 111. [ 11 ] A. Ashkin, Phys. Rev. Lett. 24 ( 1970 ) 156. [ 12 ] A. Ashkin, J.M. Dziedzic, J.E. Bjorkholm, S. Chu, Opt. Lett. 11 ( 1986 ) 288. [ 13 ] A. Ashkin, J.M. Dziedzic, T. Yamane, Nature 330 ( 1987 ) 769. [ 14 ] M.W. Berns, J.R. Aist, W.H. Wright, H. Liang, Exp. Cell. Res. 198 ( 1992 ) 375. [ 15 ] K. Nakatani, T. Uchida, H. Misawa, N. Kitamura, H. Masuhara, J. Phys. Chem. 97 ( 1993 ) 5197. [ 16 ] N. Kitamura, H.-B. Kim, S. Habuchi, M. Chiba, Trends Anal. Chem. 18 ( 1999 ) 675. [ 17 ] C. Esen, T. Kaiser, G. Schweiger, Appl. Spectrosc. 50 ( 1996 ) 823. [ 18 ] M. Trunk, J. Popp, M. Lankers, W. Kiefer, Chem. Phys. Lett. 264 ( 1997 ) 233. [ 19 ] J. Musick, J. Popp, M. Trunk, W. Kiefer, Appl. Spectrosc. 52 ( 1998 ) 692.

[ 20 ] T. Kaizer, G. Roll, G. Schweiger, J. Opt. Soc. Am. B12 ( 1995 ) 281. [ 21 ] K.D. Crawford, K.D. Hughes, J. Phys. Chem. B102 ( 1998 ) 2325. [ 22 ] B. Chase, Anal. Chem. 59 ( 1987 ) 881A. [ 23 ] K.P.J. Williams, S.M. Mason, Trends Anal. Chem. 9 ( 1990 ) 119. [ 24 ] P.J. Hendra, in: J.J. Laserna ( Editor ), Modern Techniques in Raman Spectroscopy, Chapter 3, John Wiley and Sons, West Sussex, 1996, p. 73. [ 25 ] E.E. Lawson, B.W. Barry, A.C. Williams, H.G.M. Edwards, J. Raman Spectrosc. 28 ( 1997 ) 111. [ 26 ] K. Ajito, Appl. Spectrosc. 52 ( 1998 ) 339. [ 27 ] K. Ajito, M. Morita, Surf. Sci. 427^428 ( 1999 ) 141. [ 28 ] K.P.J. Williams, G.D. Pitt, D.N. Batchelder, B.J. Kip, Appl. Spectrosc. 48 ( 1994 ) 232. [ 29 ] K. Ajito, Thin Solid Films 331 ( 1998 ) 181. [ 30 ] K. Ajito, M. Morita, Mol. Cryst. Liq. Cryst. 314 ( 1998 ) 191. [ 31 ] K. Ajito, M. Morita, K. Torimitsu, Anal. Chem. 72 ( 2000 ) 4721. [ 32 ] K. Ajito, in: S.G. Pandalai ( Editor ), Recent Research Developments in Applied Spectroscopy, Vol. 3, Research Signpost, Trivandrum, 2000, pp. 135^143. Authors are members of the Materials Science Laboratory in NTT Basic Research Laboratories, Nippon Telegraph and Telephone Corporation. Dr. Ajito obtained the B.S. and M.S. in chemistry from Keio University under the direction of Professor Masatoki Ito in 1988 and 1990, respectively. He received the Ph.D. in applied chemistry from the University of Tokyo under the direction of Professor Akira Fujishima. He joined the NTT Basic Research Laboratories in 1995. In 1999, he received the Research Paper Presentation Award from the Japan Society of Applied Physics for his paper on Raman spectroscopy of single laser-trapped droplets. He is interested in Raman microscopy, Raman imaging, laser trapping, and molecular manipulation in nanospace. Recently, he has started collaboration with Dr. Torimitsu, a neuroscientist in the same laboratory, on the study of cell organelles.