Anal. Chem. 2005, 77, 626-630
Thermal Lens Micro Optical Systems Manabu Tokeshi,† Jun Yamaguchi,‡ Akihiko Hattori,‡ and Takehiko Kitamori*,†,§
Integrated Chemistry Project, Kanagawa Academy of Science and Technology (KAST), KSP East 307, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa 213-0012, Japan, Micro Chemical Chip Project, Information Technology Company, Nippon Sheet Glass Corporation, Ltd., 5-8-1 Nishi-Hashimoto, Sagamihara, Kanagawa 229-1189, Japan, and Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan
This paper describes two types of miniaturized thermal lens optical systems that use optical fibers, SELFOC microlenses and light sources. The first system consists of a compact diode pumped solid-state laser (532 nm) as an excitation light source, a laser diode (635 nm) as a probe light source, an acoustoptic modulator as an excitation light modulator, fiber-based and conventional optics, and a detection system that combines a pinhole, an interference filter, and a photodiode. The second system consists of two laser diodes as the excitation (658 nm) and probe (780 nm) light sources, fiber-based optics, and the same detection system as the first one. The performance of the two systems was evaluated by the limit of detection (LOD) using standard solutions of sunset yellow (SY) and nickel(II) phthalocyaninetetrasulfonic acid tetrasodium salt (NiP). The LODs of the first system for SY and second system for NiP were calculated to be 3.7 × 10-8 (1.7 × 10-6 AU) and 7.7 × 10-9 M (3.4 × 10-6 AU), respectively. These results were consistent with the expected values obtained from photothermal parameters. In recent years, there has been great interest in microfluidic devices.1,2 These devices have many advantages, including simplified operations, shortened analysis time, and reduced sample, reagents, and waste quantities. These advantages are desirable for applications in a variety of fields such as (bio)analytical chemistry, clinical diagnosis, and chemical manufacturing. Making the most use of the advantages necessitates highly sensitive detection methods because the quantities of detected targets are very small. Therefore, microfluidic devices commonly employ a laser-induced fluorescence (LIF) method for detection. The LIF method has excellent sensitivity that a single molecule can be readily detected under certain conditions. Unfortunately, in principle, the LIF has a big drawback, which is the limitation of targets to fluorescent materials. Several detection methods have been proposed. Liang et al.3 developed an absorbance detection system integrated into capillary electrophoresis (CE) devices. * To whom correspondence should be addressed: E-mail: kitamori@ icl.t.u-tokyo.ac.jp. Fax: +81-3-5841-6039. † Kanagawa Academy of Science and Technology (KAST). ‡ Nippon Sheet Glass Corp., Ltd. § The University of Tokyo. (1) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (2) Auroux, P.-A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652.
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Absorbance detection methods have wide applicability because it is usually an easy task to choose a light source with a wavelength that matches the absorption band of the targets. However, for microfluidic devices, sensitivity of absorbance detection methods is often insufficient since the optical path length cannot be lengthened enough, even in the case of a multireflection configuration.4 Burggraf et al.5 demonstrated CE devices using holographic refractive index detection, and Swinney et al.6,7 developed an on-chip detection system based on backscatter interferometry. However, these have only been applied for separation systems such as CE and chromatography, and the sensitivity is still not satisfying. Therefore, the development of new chip-based detection methods with high sensitivity and wide applicability is desired. Several years ago, we discovered a photothermal effect could be generated under a microscope that was introduced coaxially into the excitation and probe laser beams.8,9 This was proved to be a thermal lens effect.10 We then developed a thermal lens microscope (TLM) having an optimized optical configuration, and we demonstrated that determination of a single-molecule level of analytes is possible under certain conditions.11 The TLM satisfies the two requirements noted above: wide applicability and high sensitivity since thermal lens spectroscopy belongs to a group of photothermal spectroscopies that rely on the measurement of heat generated from nonradiative relaxation processes followed by absorption of optical radiation and it is well known as a highly sensitive absorptiometric technique for measuring extremely low absorbances.12 However, no thermal lens spectroscopy under a microscope has been reported until we developed the TLM. Only a thermal lens microscope with cross beam configuration had been proposed.13,14 We have made several successful demonstrations (3) Liang, Z.; Chiem, N.; Ocvirk, G.; Tang, Thompson, Fluri, K.; Harrison, D. J. Anal. Chem. 1996, 68, 1040-1046. (4) Salimi-Moosavi, H.; Jiang, Y.; Lester, L.; Mckinnon, G.; Harrison, J. D. Electrophoresis 2000, 21, 1291-1299. (5) Burggraf, N.; Krattiger, B.; de Mello, A. J.; de Rooij, N. F.; Manz, A. Analyst 1998, 123, 1443-1447. (6) Swinney, K.; Markov, D.; Bornhop, D. J. J. Microcolumn Sep. 1999, 11, 596-604. (7) Swinney, K.; Markov, D.; Bornhop, D. J. Anal. Chem. 2000, 72, 26902695. (8) Harada, M.; Iwamoto, K.; Kitamori, T.; Sawada, T. Anal. Chem. 1993, 65, 2983-2940. (9) Harada, M.; Shibata, M.; Kitamori, T.; Sawada, T. Anal. Chem. Acta 1995, 299, 349-354. (10) Uchiyama, K.; Hibara, A.; Kimura, H.; Sawada, T.; Kitamori, T. Jpn. J. Appl. Phys. 2000, 39, 5316-5322. (11) Tokeshi, M.; Uchida, M.; Hibara, A.; Sawada, T.; Kitamori, T. Anal. Chem. 2001, 73, 2112-2116. (12) Bialkowski, S. E. Photothermal Spectroscopy Methods for Chemical Analysis; John Willy & Sons: New York, 1996. 10.1021/ac049011r CCC: $30.25
© 2005 American Chemical Society Published on Web 12/08/2004
of microfluidic systems using the TLM as a detection tool.15-20 The TLM has a big advantage in that the TLM signal is almost independent of the optical path length for detection using microfluidic devices. On the other hand, the size of microscope-based detection systems including light sources, microscope, optics, and detector is large relative to the size of the microfluidic devices although the miniaturization of all parts, which compose the systems, can be anticipated for various applications. From the viewpoint of miniaturization, electrochemical detection is suitable because of the ease of microfabrication and integration and light sources are unnecessary.21-23 However, the sensitivity of electrochemical detection is less than that of light detection methods such as fluorescence and thermal lens spectroscopy. Although the development of high-performance, miniaturized systems is highly desirable, reports along this line are relatively few.3,24-28 Liang et al.3 proposed a microfabricated absorbance and fluorescence detection system for CE in which optical fibers were inserted in microchannels. Webster et al.24 demonstrated a monolithic CE device with an integrated fluorescence detector. Hu¨bner et al.25 demonstrated an integrated waveguide detection system using fiber optics. Chabinyc et al.26 proposed a prototype of an integrated fluorescence detection system using a microavalanche photodiode embedded in PDMS substrate and an optical fiber. Uchiyama et al.27 demonstrated the incorporation of a blue light-emitting diode (LED) as light source into a polyester microchip for CE. Edel et al.28 reported the use of a thin-film polymer LED as an integrated excitation source for CE. All these papers are focused on fluorescence detection systems. Therefore, they cannot be applied to samples other than fluorescent materials. In this paper, we describe two types of miniaturized thermal lens optical systems using a SELFOC microlens, fiber optics, and (I) a compact laser and a laser diode or (II) laser diodes (LDs) as light sources. To demonstrate the ability of our developed systems, we have measured the thermal lens signal of nonfluorescent dye solutions and obtained calibration curves. (13) Burgi, D. S.; Nolan, T. G.; Risfelt, J. A.; Dovichi, N. J. Opt. Eng. 1984, 23, 756-758. (14) Burgi, D. S.; Dovichi, N. J. Appl. Opt. 1987, 26, 4665-4669. (15) Tokeshi, M.; Minagawa, T.; Kitamori, T. Anal. Chem. 2000, 72, 17111714. (16) Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73, 12131218. (17) Hisamoto, H.; Horiuchi, T.; Uchiyama, K.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 5551-5556. (18) Tamaki, E.; Sato, K.; Tokeshi, M.; Sato, K.; Aihara, H.; Kitamori, T. Anal. Chem. 2002, 74, 1560-1564. (19) Tokeshi, M.; Minagawa, T.; Uchiyama, K.; Hibara, A.; Sato, K.; Hisamoto, H.; Kitamori, T. Anal. Chem. 2002, 74, 1565-1571. (20) Surmerian, M.; Slyadnev, M. N.; Hisamoto, H.; Hibara, A.; Uchiyama, K.; Kitamori, T. Anal. Chem. 2002, 74, 2014-2020. (21) Woolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1998, 70, 684-688. (22) Zhao, H.; Dadoo, R.; Reay, R. J.; Kovacs, G. T. A.; Zare, R. N. J. Chromatogr., A 1998, 813, 205-208. (23) Rossier, J. S.; Roberts, M. A.; Ferrigno, R.; Girault, H. H. Anal. Chem. 1999, 71, 4294-4299. (24) Webster, J. R.; Burns, M. A.; Burke, D. T.; Mastrangelo, C. H. Anal. Chem. 2001, 73, 1622-1626. (25) Hu ¨bner, J.; Mogensen, K. B.; Jorgensen, A. M.; Friis, P.; Telleman, P.; Kutter, J. P. Rev. Sci. Instrum. 2001, 72, 229-233. (26) Chabinyc, M. L.; Chiu, D. T.; McDonald, J. C.; Stroock, A. D.; Christian, J. F.; Karger, A. M.; Whitesides, G. M. Anal. Chem. 2001, 73, 4491-4498. (27) Uchiyama, K.; Xu, W.; Qiu, J.; Hobo, T. Fresenius J. Anal. Chem. 2001, 371, 209-211. (28) Edel, J. B.; Beard, N. P.; Hofmann, O.; deMello, J. C.; Bradley, D. D. C.; deMello, A. J. Lab Chip 2004, 4, 136-140.
Figure 1. Schematic illustration of optical configuration for thermal lens microscope: (a) proper and (b) unsuitable configuration.
THERMAL LENS OPTICAL SYSTEMS Principle of Thermal Lens Measurement. Figure 1 shows a schematic illustration of the optical configuration required for thermal lens microscopy. In thermal lens microscopy, it is very important to shift the focal points of the excitation and probe beams.10,11 If the locations of the focal points of two beams are the same as shown in Figure 1b, the probe beam is not affected very much by a thermal lens induced by the excitation beam. Thus, the sensitivity in this case is very low. To realize high sensitivity, the locations of two focal points must be shifted as shown in Figure 1a. However, although a commercially available ordinary objective lens has an aplanat, i.e., the locations of two focal points become the same when two beams are introduced into the objective lens, highly sensitive thermal lens measurements cannot be realized. Actually, early in the development of our TLM, we used an old-type objective lens with chromatic aberration.8-10 Presently, our TLM uses an ordinary objective lens and focal pointadjustable optics.11 By adjusting this optics, the location of the focal point of the probe beam can be moved arbitrarily and the relative distance between foci of the excitation and probe beams can be optimized for highly sensitive thermal lens measurements.11 Characteristics of (GRadient Index) (GRIN) Microlens. As described above, the most important consideration for the TLM measurements is the difference between the focal points of the excitation and probe beams. This must be dealt with at the beginning when we construct a miniaturized thermal lens optical system. We looked at a gradient index microlens. The gradient index microlens, known commercially as a GRIN lens, is used widely in optical communication systems. Its features are as follows: (i) It has a gradient refractive index within the lens. (ii) It has a cylindrical rod shape. (iii) Generally the end faces of the lens are flat. (iv) It is quite easy to connect and collimate the lens with optical fibers. Of course, the GRIN lens has chromatic aberration. Moreover, the location of the focal point can be adjusted arbitrary by changing either the ion-dope quantity in the rod or the length of the lens. Basically, by combining lenses of two different rod lengths, it is also possible to optimize the locations of the focal points of the excitation and probe beams for thermal lens measurements. As described earlier, these features are well suited to construction of miniaturized thermal lens optical systems. Analytical Chemistry, Vol. 77, No. 2, January 15, 2005
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Figure 2. Schematic of the thermal lens optical system using a compact laser and a laser diode (system I).
Figure 3. (a) Schematic and (b) photograph of the SELFOC lens supported by a lens holder. The single-mode optical fiber is held by a capillary.
EXPERIMENTAL SECTION Thermal Lens Optical System I Using Compact Lasers. Figure 2 shows a schematic of the thermal lens optical system using a compact laser and a laser diode. The excitation beam was the 532-nm emission line of a diode pumped solid-state laser (CrystaLaser, Reno, NV, model GCL-20-S) with output power of 20 mW, which was modulated by an acoustoptic modulator (HoyaSchott Co., Tokyo, A-140) at 1 kHz. A laser diode (Hitachi Ltd., Tokyo, Japan, HL6321G, 5 mW) with an emission line of 635 nm was used for a probe beam. The two beams were made coaxial by a dichroic mirror and introduced into a single-mode optical fiber (FiberCore Inc., Charlton, MA, outer diameter 125 µm, core diameter 4 µm). The end of the optical fiber was equipped with a GRIN lens, which was used to focus the beams. In the present study, the SELFOC lens (Nippon Sheet Glass Corp. Ltd., Tokyo, Japan, SLC10-025p, NA 0.2) was used as a GRIN lens and it had a diameter of 1 mm and length of 2.3 mm. The SELFOC lens was supported by a lens holder (Figure 3). The distance between the focal points of the two beams was 35 µm. The powers of the excitation and probe beams just below the SELFOC lens were 3.4 and 0.22 mW, respectively. The two focused beams were irradiated into a sample cell (GL Sciences Inc., Tokyo, Japan, AB628
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20), and the thermal lens effect in the sample cell was induced by the periodically modulated excitation beam. The focal point of the probe beam was changed by the thermal lens effect. The probe beam passed through an interference filter (Edmund Optics Japan Co. Ltd., Tokyo, Japan, 34082-F) and a pinhole of 1-mm diameter before being detected by a photodiode (Hamamatsu Photonics K.K., Hamamatsu, Japan, S1337-33BR). The electrical signal from the photodiode was fed into a lock-in amplifier (NF Corp., Yokohama, Japan, LI-575). The synchronous signal with modulation frequency was acquired as a thermal lens signal using a PC via a GP-IB card (National Instruments, Austin, TX) and a custom LabView program (National Instruments). Thermal Lens Optical System II Using Laser Diodes. Figure 4 shows a schematic of the thermal lens optical system using the LDs. The main differences between this and the former one were the use of a LD as an excitation light source, a wavelength division multiplexing (WDM)-type optical multiplexer, and application of the power supply modulation method in place of a compact laser, a dichroic mirror, and a light modulator, respectively. The detection system including an interference filter, a pinhole, and a photodiode was the same as in the former system. The excitation beam was the 658-nm emission of one LD (Hitachi Ltd., HL6501MG, 35 mW), which was modulated by using the power supply modulation method at 1 kHz. A near-infrared 785nm emission line of the other LD (Hitachi Ltd., 7859MG, 35 mW) was used for a probe beam. These two beams from the LDs were introduced into the single-mode optical fibers using the GRIN lenses. Then, the two beams were multiplexed by a WDM-type multiplexer (Nippon Sheet Glass Ltd., custom-made). The multiplexer consisted of two SELFOC lenses (Nippon Sheet Glass Ltd., SLC18-025p). The end surface of each lens was coated with a dielectric multilayer (Nippon Sheet Glass Ltd., custom-made, transmittance >90%, reflectance >90%). The excitation beam was reflected by the dielectric multilayer and was introduced into another single-mode optical fiber. The probe beam was transmitted through two lenses and was introduced into the same optical fiber as the excitation beam was introduced into. Finally, the two beams were focused onto the liquid sample in the sample cell by a SELFOC lens that was connected with the end of the fiber. The difference between the focal points of the two beams was 37 µm. The powers of the excitation and probe beams after passing
text samples. For the thermal lens optical system I, the dependence of the thermal lens signal intensity on the concentration of SY aqueous solutions was measured. There was good linearity in the range of 1 × 10-7-1 × 10-4 M. Using the results below 1 × 10-6 M (with a correlation efficient of 0.9837), we estimated the determination limit, by doubling the standard deviation 2σ, and the detection limit, S/N ) 2, to be 1.5 × 10-7 and 3.7 × 10-8 M, respectively. For the thermal lens optical system II, the dependence of the thermal lens signal intensity on the concentration of NiP aqueous solutions was obtained. There was also good linearity in the range of 1 × 10-8-1 × 10-5 M. Using the results below 1 × 10-7 (with a correlation efficient of 0.9994), we estimated the determination limit (2σ) and the detection limit (S/N ) 2) to be 6.5 × 10-8 and 7.7 × 10-9 M, respectively. For the performance evaluation of these two optical systems, it is necessary to take into account the power of the excitation light and molar extinction coefficient of the sample at the excitation wavelength, since the thermal lens signal, STL, can be expressed with the following formula.12
STL ) EA )
Figure 4. (a) Schematic of the thermal lens optical system using the LDs (system II). (b) Photograph showing the appearance of the single box (the part of the dashed line in (b)). (c) Schematic of the WDM-type multiplexer.
through the SELFOC lens were 2.2 and 0.61 mW, respectively. The two beams passing through the sample cell were introduced into the same detection system as in system I expect for use of an interference filter (Edmund Optics Japan Co. Ltd., 43093-F). Chemicals. The samples were aqueous solutions of two dyes, sunset yellow (SY) (532 ) 4700) and nickel(II) phthalocyaninetetrasulfonic acid, tetrasodium salt (NiP) (658 ) 43700) from Wako Pure Chemical Industries (Osaka, Japan) and Aldrich Chem. Co. (Milwaukee, WI), respectively, and were used as-received. Stepwise dilution of stock solutions was used to get sample solutions. Measurements. A quartz demountable cell (GL Sciences, Inc., AB20) was used as a sample cell. This cell had an optical path length of 100 µm. We confirmed that the same signal intensity of the thermal lens was obtained with this cell and a microchannel (width 250 µm, depth 100 µm) that was purchased from the Institute of Microchemical Technology (Kawasaki, Japan) when using the two systems developed here. To simplify the experimental procedures, we used the demountable cell in place of the microchip. RESULTS AND DISCUSSION To evaluate the performance of the thermal lens optical systems, we performed the thermal lens measurement using the
Pe(dn/dT)A λpκ
(1)
Here, E ()Pe(dn/dT)/λPκ) is the thermal lens enhancement factor, A ()Cl, where is the molar absorption coefficient, C is the concentration of the solution, and l is the path length of the cell) is the absorbance, Pe is the power of the excitation light, dn/dT is the refractive index gradient, λP is the wavelength of the probe light, and κ is the thermal conductivity of the solvent. Since the dn/dT and κ are the same between the experiments using systems I and II, only the magnitude of the Pe, A, and λP affects the STL. The optical parameters of the systems and samples and the detection limits by systems I and II are summarized in Table 1. The difference between the detection limits can be explained well when the differences of Pe, , and λP are taken into consideration. In other words, the measurements using system II should be (PeIINiPλPI/PeISYλPII) times better than those of system I, where PeI and PeII are the powers of the excitation light of systems I and II, NiP and SY are the molar extinction coefficient of NiP and SY at the excitation wavelength and λPI and λPII are the wavelengths of the probe light of systems I and II, respectively. Actually, STL was ∼4.9, and this agreed with the difference in the detection limits between the two systems. Of course, Pp and Def (Table 1) and other optical parameters are also related to the detection limit.12,29 However, in the present work, the differences in these parameters between two systems could be neglected. Next, the performance of the systems developed here was compared with that of the TLM.30,31 The detection limit, photothermal parameters, and molecular properties of samples from the present and previous studies are shown in Table 2. Since the experimental parameters of system I were almost the same as those of TLMYAG, system I and TLMYAG were compared. As seen from Table 2, for the two, the only difference in the photothermal parameters in formula 1 was the power of the excitation light, Pe, since the difference in the wavelength of probe light, λP, was (29) Proskurnin, M. A.; Slyadnev, M. N.; Tokeshi, M.; Kitamori, T. Anal. Chim. Acta 2003, 480, 79-95. (30) Hiki, S.; Tokeshi, M.; Hibara, A.; Kitamori, T. Bunseki Kagaku 2003, 52, 569-574 (in Japanese). (31) Hiki, S.; Tokeshi, M.; Kitamori, T. Unpublished data.
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Table 1. Parameters for Thermal Lens Measurement and the Detection Limits
system i system ii
Pe (mW)a
Pp (mW)a
(M-1 cm-1)b
numerical aperture
λP (nm)
Dep (µm)c
detection limit (M)
detection limit (AU)
3.4 2.2
0.22 0.61
4700 43700
0.2 0.2
635 785
35 37
3.7 × 10-8 7.7 × 10-9
1.7 × 10-6 3.4 × 10-6
a The power of the excitation beam below the SELFOC lens. b The power of the probe beam below the SELFOC lens focal points of the excitation and probe lights.
c
Difference between the
Table 2. Photothermal Parameters and Molecular Properties
present work system I system II previous work TLMAr TLMYAG TLML
detection limit (M)
Pe (mW)a
Pp (mW)b
dn/dT (K-1 × 104)c
λE (nm)
water water
3.7 × 10-8 7.7 × 10-9
3.4 2.2
0.22 0.61
-0.91 -0.91
532 658
water water benzene
1.8 × 10-9 2.3 × 10-8 7.4 × 10-11
20 9.3 2
1 1 0.1
-0.91 -0.91 -6.52
488 532 488
solute
solvent
SY NiP SY SY OEP
λP (nm)
κ (W m-1 K-1)c
(M-1 cm-1)d
635 785
0.598 0.598
4700 43700
632.8 632.8 632.8
0.598 0.598 0.137
22400 4700 32000
a The power of the excitation beam below the SEFLOC lens. b The power of the probe beam below the SEFLOC lens. c dn/dT and k data from ref 12. d The molecular absorption coefficient of sample of the excitation wavelength.
negligible. The Pe of TLMYAG was ∼2.7 times larger than that of system I. The LOD of TLMYAG was ∼1.3 times better than that of system I. This slight difference may be caused by the experimental conditions, i.e., electric noise, mechanical vibration, etc. When system II was compared with that of TLMAr, the difference between them can be described as
frequency of the excitation light, the shape of the sample cell, the excitation-light cut filter, and so on differed from those of the others. These differences should have an effect on the LOD. Although the performance of the systems developed here was a little lower than that of TLML, they still had very good performance despite miniaturization.
PeTLMTLM/λPTLM PeIIII/λPII
CONCLUSIONS AND PERSPECTIVES We have described the miniaturization of thermal lens detection systems based on an optical fiber and SELFOC microlens. The systems developed here showed good performance for detection of nonfluorescent molecules despite the miniaturization of optical components. In the future, these systems lead to chipbased compact analysis systems incorporating a detection system. Practically, we are in the progress of developing a chip-based enzyme-linked immunosorbent assay system now. Moreover, we are wrestling with the multiplexation of a thermal lens detection system to detect multisamples simultaneously. Furthermore, we want to try to change the wavelength of the excitation laser diode in order to expand application areas.
(2)
where PeII and PeTLM are the powers of the excitation light of system II and TLMAr, II, and TLM are the molar absorption coefficients of NiP at 658 nm and SY at 532 nm, λPII and λPTLM are the wavelengths of the probe light of system II and TLMAr, respectively. The value calculated by using these parameters was ∼5.8. The LOD of TLMAr was ∼4.3 times lower than that of system II. This result was quite reasonable. The present work had a slightly better performance than was previously obtained with TLM. Using the TLM and a benzene solution of Pb(II) octaethylporphyrin (OEP), we succeeded in obtaining the LOD of 7.4 × 10-11 M.11 This value is the lowest LOD that has been obtained with TLM. The photothermal parameters when the lowest LOD was obtained are also shown in Table 2. The previous experimental conditions are also represented by TLML. The performance of system II was compared with that of TLML by considering the photothermal parameters as done above. Although the performance of TLML was ∼25.8 times better than that of system II, the LOD of TLML was ∼104.1 times lower than that of system II. Therefore, the performance of TLML was 4 times better than that of system II. This may be due to the experimental conditions. The experimental conditions for all systems in Table 2 except TLML were almost the same. But for TLML, in the case of the TLML, the experimental conditions such as the modulation
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Note Added after ASAP Publication. A minor change was made to the title and the paper was reposted on 12/29/04. ACKNOWLEDGMENT We thank Dr. Akihide Hibara of The University of Tokyo, Mr. Takashi Fukuzawa and Mr. Yoshinori Matsuoka of Nippon Sheet Glass Co., Ltd., and Mr. Shinichiro Hiki of the Institute of Microchemical Technology for valuable discussions. We also acknowledge Miss Hiroko Takahashi of KAST for taking the photographs used in this article. Received for review July 6, 2004. Accepted October 25, 2004. AC049011R
