near-infrared spectroscopy of single particles

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biological scientists, is that it allows the manipula-tion of not only cells but also of organelles and

vesicles within cells [ 13,14].The optical tweezers have been used with spec-

troscopic techniques to characterize moleculescontained in single particles. Chemical reactions

within single microdroplets or microcapsules

have been studied using systems that combinelaser trapping with electrochemistry [ 15 ], £uores-cence spectroscopy [ 15,16], absorption spectros-copy [ 16], or Raman spectroscopy [ 17^19].Raman spectroscopy is advantageous as an analyt-ical tool for trapped particles because it providesinformation about species, structures, and confor-mations of various kinds of molecules in the par-ticle. Another advantage regarding instrumentationfor Raman measurements is that the focused laser beam can be used for both laser trapping andRaman spectroscopy.

Visible laser light has been widely used as anexcitation light source for Raman spectroscopy tostudy 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 typicalRaman spectroscopic technique using NIR laser light, is dif¢cult with conventional optical micro-scopes and has a much lower sensitivity than visibleRaman spectroscopy. Nevertheless, a highly sensi-tive Raman spectroscope using NIR laser light isrequired in microchemistry, mesoscopic chemistry,and biological engineering for analyses of trapped

particles, because the NIR laser light eliminates £u-orescent background in the Raman spectra of organic and biological materials. Recently, wedeveloped a new NIR Raman microscope compris-ing a high-power NIR laser, an optical microscope,holographic notch ¢lters (HNFs), a single-gratingpolychromator, and a charge-coupled device(CCD) camera. Then, we demonstrated a systemthat combined NIR Raman microscopy with thelaser trapping technique, which is called theRaman 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 trapsingle particles without damaging them and pro-

vides a wealth of molecular information about those particles. The paper presents the recent results of a RTM analysis of tiny particles and alsodiscusses the differencein behavior between a mol-ecule 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 andRaman spectroscopy in the system. The laser trap-ping technique employs the force of gradient radi-ation pressure generated when a laser beam is

tightly focused onto a small particle under an opti-cal microscope. Although small particles in solutionare always moving by Brownian motion, they canbe 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 par-ticle and its momentum is changed. The change of the momentum causes the force of radiation pres-sure to pull the particle toward the beam (shown inthe particle model illustrated in Fig. 1). Focus thebeam beyond the center of the particle and the par-ticle will be pushed away, while a focal point to theleft or right of center would cause the particle tomove left or right. Consequently, the particle iscompletely trapped by the optical force.

The experimental apparatus is described brie£y here. The laser light source is a continuous-wave,single-frequency titanium:sapphire laser (Titan-Cw, Schwartz Electro-Optics) tuned from 730 to780 nm in the TEM00 mode. The pump source for the titanium:sapphire laser is the 532-nm line of asolid-state continuous-wave laser (Millenia, Spec-tra-Physics Lasers). A commercial Raman microp-robe spectrometer (Ramascope, Renishaw) wasspecially modi¢ed for NIR laser light. The system

is controlled by a Windows-type personal com-puter. The expanded NIR beam is focused ontothe sample using an objective lens with 100Umag-ni¢cation and a numerical aperture of 0.8. The lensis mounted on an optical microscope ( BH-2, Olym-pus). The objective lens used to focusthe laser ontothe sample is also used to collect light scatteredfrom the sample at 180³ with respect to the incident light. After the scattered light passes through twoHNFs to remove Rayleigh scattered light, it isfocused onto the entrance slit of a single-gratingpolychromator, which is then focused onto a CCD

camera ( 02-06-1-225, Wright Instruments). ThisCCD camera contains a Peltier-cooled slow-scan384U576 CCD chip maintained at V200 K.

In the original system, the CCD camera that records a Raman spectrum was also used to obtainan optical image of a trapped particle [ 26 ]. How-ever, in the new version, the microscope was ¢tted

with an additional CCD camera and a HNF [ 27 ]. A

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



dielectric multilayer-coated beam splitter in themicroscope divides the scattered NIR light intotwo paths, one for each CCD camera. The two-slit confocal arrangement [ 28^30] is used to eliminateRaman scattered light from the outer region of theparticle. The ¢rst slit is the entrance slit of polychro-matorand thesecond slit is the very narrow readout

window for the CCD camera. The slits are perpen-dicular to each other. Using these slits instead of pinholes makes it easier to make the optical align-ments needed for confocal Raman measurements.

3. Experiments and results

3.1. Raman spectroscopy of a trapped droplet

The RTM enables us to obtain Raman spectrafrom a single tiny droplet. Fig. 2 illustrates the opti-cal arrangement for a toluene droplet in a hemi-

spherical water drop ¢xed in the sample cell [ 26 ].Toluene was selected as the solvent for dropletsbecause it has a much larger refractive index N D(N D=1.497 at 293 K) than water (N D=1.333 at 293K ). The higher N D leads to a larger optical radiationforce in water. The droplets were made from a tol-uene^water mixture by an ultrasonic treatment.The toluene droplets gradually aggregated at thecenter of the water drop at the surface. Therefore,the laser trapping was carried out at the water dropsurface away from the area where the droplets gath-ered.Thelaserspotfocusedusingtheobjectivelens

wasV1 Wm indiameter ata power ofabout 80mW. A single toluene droplet about 15 Wm in diameter was trapped in the vertical direction almost imme-diately and was completely trapped in the lateraldirection within 20 s after laser illumination wasstarted. The Raman spectrum for the single trappedtoluene droplet was clearly obtained below 100cm31 to above 3000 cm31 as shown in Fig. 2. The

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

trends in analytical chemistry, vol. 20, no. 5, 2001 257



total exposure time in this scanning range was 5 s.Noteworthy is the absence of £uorescence interfer-

ence in the Raman spectrum due to the use of NIR laser light. Furthermore, the signal-to-noise ratio of the spectrum is suf¢cient to identify the molecular species of a single droplet.

ARaman spectrum obtained by using RTM can beapplied to the quantitative analysis of a singletrapped particle [ 27 ]. Sample droplets were madefrom a p -cresol toluene mixture with ultrasonictreatment in deionized water and mainly rangedfrom 10 to 20 Wm in diameter. The image in Fig. 3shows the droplets in water under the objectivelens. One droplet was trapped by the laser probe

(upper right) and the other droplet was free andmoving by Brownian motion (lower left). Thebright spot in the trapped particle is the focal spot of the laser beam at a power of 120 mW, visualizedusing the additional CCD camera. The intensity of the NIR light re£ected from the focused spot ismuch higher than the white light re£ected fromthe 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.

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.




sity of the scattered NIR light by a factor of about 1034, which prevents CCD pixel saturation.

The Raman spectrum of the trapped droplet isspectrum A in Fig. 4. The exposure time of thisRaman spectrum was 3 s. The relative intensity among peaks in a Raman spectrum gives us theconcentration ( the mole fraction) of each species.Spectrum B is the Raman spectrum for the bulk

solution, which was made by mixing p -cresol (30mol%) and toluene ( 70 mol%). It was also obtainedin the exposure time of 3 s. The peaks at 785, 1003,and 1030 cm31 are attributed to the ring-breathingmodeoftolueneandthepeaksat823and843cm31

are attributed to the doublet of the ring-breathingmode of p -cresol. These peaks have been observedfor a large number of para -substituted benzenes.The spectra were normalized with the toluenepeak at 1003 cm31. The intensities of the peaksfor 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, theconcentration of p -cresol calculated form the spec-trum is V7 mol% in the droplet. This result indi-cates the RTM system can easily determine the con-centration of each molecular species in singleparticles.

3.2. Molecular extraction in single droplets

Fig. 5 shows our recent results concerning theliquid^liquid extraction process in a single subpico-liter droplet [ 31 ]. Fig. 5A shows the set of the time-dependent images of a trapped single subpicoliter droplet in water. The four images are of the same

area. Theimage at the far left shows a single toluenedroplet just after it was trapped. The trapped drop-let isV10 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 isthe focal point ofthe laserbeam.The other imagesshow the trapped droplet 1, 2, and 3 min after p -nonylphenol (PNF) was added to the solution.

Fig. 5B shows the time-dependent Raman spec-tra of the trapped droplet in the images in Fig. 5A.The exposure time for each spectrum was 3 s. Thesharp peaks at 785, 1003, 1030 cm31 are attributedto the modes of the phenyl group of toluene. Thefour spectra were normalized by using the peaks of tolueneat1003cm31.OneminuteafterthePNFwasadded, 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 normalizedpeaks of toluene and their intensities saturated after that. These results indicate that the trapped droplet extracted PNF from the solution, which caused thedroplet to increase in size as shown in Fig. 5A.

The same experiment was done nine more timesfor droplets of various sizes. Fig. 5C shows the initial

droplet size dependence of the concentration andthe distribution coef¢cient of PNF in single subpico-liter and picoliter droplets. The distribution coef¢-cient is de¢ned as the ratio of PNF concentration inthe droplet to that in the solution around the drop-let. The dashed line indicates the distribution coef-¢cient of PNF in bulk solution. The result indicatesthe liquid^liquid extraction process in dropletsranging from subpicoliter to picoliter is different from that in bulk solution and the difference is

very large for a subpicoliter droplet. This phenom-enon can be explained considering the droplet sur-

face areas. The ratio of the surface area to the vol-ume of a subpicoliter droplet is much larger thanthat of solution in a conventional container, such asa separating funnel, and this strongly affects thesurface reactions in the liquid^liquid extractionprocess.

Fig. 4. NIR Raman spectra of the single trapped droplet (A)

and bulk solution (B).




3.3. Raman spectroscopy of smaller single particles

The RTM system can be applied for the analysisof a smaller particle [ 32]. The smallest droplet in theexperiments described above was V10 Wmbecause it is dif¢cult to make toluene droplets any smaller than that. The laser probe of the RTM sys-tem has about a 1- Wm diameter at the focal plane,

which makes it possible to obtain a Raman spec-trum from a single particle in the several micro-meter range without loss of sensitivity. In this sec-tion, the Raman measurement of smaller polymer spheres is shown.

Image B in Fig. 6 shows a single polymer spheretrapped in water. The trapped sphere is a polystyr-ene latex bead about 2.1Wm in diameter. The refrac-

tive index of the beads is 1.580 (at 293 K), which islarger than toluene and makes them easier to trap in

water. Image A was recorded in the same area asimage B before trapping. Image B shows the singlebead trapped in water. There are no other particlesin the image because the sample solution was very dilute. The spectra corresponding to these imagesare also shown in Fig. 6. The exposure time for each

spectrum was 5 s. The spectrum corresponding toimage A shows the background of the solution andthe spectrum corresponding to image B shows theRaman spectrum of the single trapped polymer sphere. The signal-to-noise ratio in the spectrumof the single trapped particle is suf¢cient to allow identi¢cation of the molecular species of the par-ticle. These results indicate the system can trap sin-

Fig.5. Time-dependentimages of a single subpicoliter toluenedroplet during liquid^liquid extraction of PNF( A) andthe NIRRaman

spectra corresponding to their images ( B ). Initial droplet size dependence of the concentration and the distribution coef¢cient for thesingle subpicoliter andpicoliter droplets C. The dashed line in C indicates the distribution coef¢cientof PNF in the bulk solution.




gle particles with diameters as small as severalmicrometers and provide their Raman spectra.

The detection of particles in the submicrometer range, the size range of many kinds of organellesin cells, will be very important in biological engi-neering. However, it is necessary to increase thesensitivity of the system for such very small particlesbecause the sensitivity decreases with decreasingparticle size.

4. Conclusions

The RTM was developed for the characterization

of organic and biological single particles. The RTMsystem, which combines NIR Raman spectroscopy

with the laser trapping technique, provides Ramanspectra of single trapped particles in a micrometer range and makes it possible to determine the par-ticles' chemical compositions, such as their molec-ular species and structures. The advantage of the system is that it uses NIR laser light instead of

visible laser light, which prevents photochemicaldamage to organic and biological particles during

laser trapping and results in much lower £uores-cence interference in the Raman spectra of trappedparticles. Our recent experiment showed that themolecular behavior in a single droplet is different from that in bulk solution during the liquid^liquidextraction process, which is due to the restrictionof molecular diffusion by the surface of the drop-let. With further improvements, the RTM willbe able to reveal the features of molecules insingle nanoparticles and will open the way to theanalysis of biological molecules in very smallorganelles. 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. Takaya-nagi (NTT Basic Research laboratories) for their

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




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

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Authors are members of the Materials Science Laboratory in NTT Basic Research Laboratories, Nippon Telegraph and TelephoneCorporation. Dr. Ajito obtained the B.S. and M.S. in chemistry from Keio University under the direction of Professor Masatoki Itoin 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 ResearchLaboratories 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, hehas started collaboration with Dr. Torimitsu, a neuroscientist in the

same laboratory, on the study of cell organelles.


near-infrared spectroscopy of single particles

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