In Situ Detection of Birefringent Mesoscopic H and J Aggregates of

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Langmuir 2005, 21, 1067-1073

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In Situ Detection of Birefringent Mesoscopic H and J Aggregates of Thiacarbocyanine Dye in Solution Hiroshi Yao,* Kaori Domoto, Takeshi Isohashi, and Keisaku Kimura Graduate School of Material Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan Received August 23, 2004. In Final Form: October 28, 2004 Polarized-light microscopy, fluorescence microscopy, atomic force microscopy as well as absorption and fluorescence spectroscopy were used to characterize mesoscopic structures of both supramolecular H and J aggregates of 3,3′-disulfopropyl-5,5′-dichloro-9-methyl thiacarbocyanine dye in aqueous solution. Polarizedlight microscopy visualizes in situ the mesoscopic morphology of the H and J aggregates and distinguishes between them by their own colors. The H aggregate having a fibrous structure showed negative birefringence, namely, the refractive index along the fiber short axis was higher than that of the long axis, so that π-electron chromophores of the dye molecule are likely to orient along the short axis of the elongated fibers. The degree of birefringence of the H aggregate fiber was ∼-0.3. Investigations on the concentration dependence of the absorption spectra showed that the amount of J aggregates increased at the expense of a decrease in the amount of H aggregates. With respect to the J aggregates, a small dot morphology was observed at a relatively low dye concentration of 3.0 mM. With an increase of the dye concentration up to 10 mM, the morphology changed into mesoscopic fibers. In contrast, fluorescence microscopy for the fibrous J aggregates reveals that the constituent molecules are approximately aligned along the long axis of the fibers.

Introduction Molecular aggregation into well-defined supramolecular structures plays a significant role in biology and in molecular electronics, and thus it has attracted a great deal of attention in both fundamental and applied research.1 In biological photosynthetic systems, lightharvesting occurs in stacked protein-bound chlorophyllcarotenoid complexes, where the antenna pigments with one-dimensional circular aggregates of molecules transfer captured sunlight energy to the reaction center as efficiently as possible.2 In molecular devices, memory or information transmission processes are dominated by characteristic collective properties of the aggregates related to the coherency of their excited states.3 Many extended supramolecular assemblies are constructed through various noncovalent interactions such as hydrogen-bonding, coordination, hydrophobic, electrostatic, and π-π stacking interactions;4 therefore, elucidation and controls of such supramolecular structures are desirable. For supramolecular aggregates that are enthalpically induced via dipole-dipole interactions between chromophores, the optical properties differ remarkably from those of single molecules and pure crystals, and thus, specific attention has been paid to clarify the spectroscopic nature theoretically. It has been established that the change in spectroscopic properties upon aggregation originates from intermolecular excitonic interactions in the aggregate that couple the optical transitions of * Corresponding author. E-mail: [email protected]. (1) Alivisatos, A. P.; Barbara, P. F.; Castleman, A. W.; Chang, J.; Dixon, D. A.; Klein, M. L.; McLendon, G. L.; Miller, J. S.; Ratner, M. A.; Rossky, P. J.; Stupp, S. I.; Thompson, M. E. Adv. Mater. 1998, 10, 1297. (2) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. A.; Cogdell, R. J.; Isaacs, N. W. Nature 1995, 374, 517. (3) Fabian, J.; Nakazumi, H.; Matsuoka, M. Chem. Rev. 1992, 92, 1197. (4) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1991.

individual molecules, resulting in the formation of two different types of aggregates:5,6 one with a red-shifted and narrow-banded absorption compared to the monomer absorption band, known as J aggregates or Scheibe aggregates,7,8 and the other with a blue-shifted absorption band, called H aggregates.5 It is well-known that J aggregates are often used as photographic sensitizers.9 Recent interest has focused on the ability of both J and H aggregates to exhibit cooperative emission phenomena such as superradiance.10,11 When molecules (or chromophores) are parallel aligned, two new excitonic bands are generated according to a simple exciton theory: one with higher energies and the other with lower than the monomer energy level. In J aggregates, transitions only to the low energy states of the exciton band are allowed, and as a consequence, J aggregates are characterized by little Stokes-shifted fluorescence that has a high quantum yield. In contrast, H aggregates are characterized by a large Stokes-shifted fluorescence with a very low quantum yield, because the excited energy to the high energy state of the H band rapidly relaxes within the excitonic band down to states with vanishing transition dipole moments.5,12 The transition energy of such aggregates is explained by the differences in the geometry of the molecular packing within the aggregates,12 and thus, these aggregates are structurally distinguished by the different slip angles between the molecular transition moment and the long axis of the (5) Wang, M.; Silva, G. L.; Armitage, B. A. J. Am. Chem. Soc. 2000, 122, 9977. (6) McRae, E. G. Aust. J. Chem. 1961, 14, 229; 344. (7) Jelley, E. E. Nature 1936, 138, 1009; 1937, 139, 631. (8) Scheibe, G. Angew. Chem. 1936, 49, 563; 1937, 50, 212. (9) Herz, A. H. In The Theory of the Photographic Process, 4th ed.; James, T. H., Ed.; Macmillan: New York, 1977. (10) (a) De Boer, S.; Wiersma, D. A. Chem. Phys. Lett. 1990, 165, 45. (b) Spano, F. C.; Kuklinski, J. R.; Mukamel, S. J. Chem. Phys. 1991, 94, 7534. (11) Meinardi, F.; Cerminara, M.; Sassella, A.; Bonifacio, R. Tubino, R. Phys. Rev. Lett. 2003, 91, 247401. (12) Kobayashi, T., Ed. J-Aggregates; World Scientific: Singapore, 1996.

10.1021/la0479004 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/14/2004

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aggregatesa small slip angle for the J aggregates and a large slip angle for the H aggregates. Despite the multitude of studies available on spectroscopic properties of such aggregates, the knowledge of their supramolecular structures is still fragmentary, and the relationship between molecular arrangements and mesoscopic properties is not clear.13-16For J aggregates, however, recent progress has specifically exhibited direct observations or visualization of the mesoscopic structures. Cryo-transmission electron microscopy (cryo-TEM) is proved to be a useful method to allow direct observation of morphologies and supramolecular structures of J aggregates, and a fiber or fiber-bundle structure of the pseudoisocyanine (PIC) J aggregate has been identified.17-19 Note that it cannot distinguish dynamic processes of the supramolecular aggregates in situ. In contrast, our previous studies based on fluorescence microscopy and atomic force microscopy (AFM) have revealed dynamic behaviors of thiacyanine J aggregates that showed spontaneous morphology transformation from a mesoscopic string structure into a tubular rod shape in aqueous solution.20-22 For H aggregates, despite the pioneering work by Emerson et al. that demonstrated a mesoscopic morphology of thiacarbocyanine H aggregates by ex situ TEM measurements,23 the knowledge of the mesoscopic structure is still rather poor because of their few observations. In the case of the solution-phase H aggregates of PIC, the assemblies of mesoscopic size have so far never been observed by the microscopic techniques,19 whereas the results of static light scattering implied the presence of nanoscopic particles of H aggregates.24 The contradiction would arise from the differences between in situ and ex situ observations. Fluorescence microscopy, although it has been an excellent method for the direct observation of mesoscopic J aggregates,20,21 cannot provide significant information on the morphology of H aggregates because of their low quantum yield of fluorescence. Hence the direct and in situ observation for the H aggregates is desirable. Polarized-light microscopy is known to exploit optical anisotropy to reveal detailed information about the birefringent structure. We have characterized birefringent properties of mesoscopic thiacyanine J aggregates using polarized-light microscopy, because the J aggregate possesses optical anisotropy caused by a well-ordered molecular alignment within the aggregate.22 We consider that, if the H aggregate grows to form mesoscopic-sized supramolecular assemblies having regular molecular alignments with strong birefringence, the aggregate also (13) Janssens, G.; Touhari, F.; Gerritsen, J. W.; van Kempen, H.; Callant, P.; Deroover, G.; Vandenbroucke, D. Chem. Phys. Lett. 2001, 344, 1. (14) Higgins, D. A.; Kerimo, J.; Vanden Bout, D. A.; Barbara, P. F. J. Am. Chem. Soc. 1996, 118, 4049. (15) Yao, H.; Sugiyama, S.; Kawabata, R.; Ikeda, H.; Matsuoka, O.; Yamamoto, S.; Kitamura, N. J. Phys. Chem. B 1999, 103, 4452. (16) Owens, R. W.; Smith, D. A. Langmuir 2000, 16, 562. (17) von Berlepsch, H.; Bo¨ttcher, C.; Ouart, A.; Burger, C.; Da¨hne, S.; Kirstein, S. J. Phys. Chem. B 2000, 104, 5255. (18) von Berlepsch, H.; Bo¨ttcher, C.; Da¨hne, L. J. Phys. Chem. B 2000, 104, 8792. (19) von Berlepsch, H.; Bo¨ttcher, C. J. Phys. Chem. B 2002, 106, 3146. (20) Yao, H.; Omizo, M.; Kitamura, N. Chem. Commun. 2000, 739. (21) Yao, H.; Kitamura, S.; Kimura, K. Phys. Chem. Chem. Phys. 2001, 3, 4560. (22) Yao, H.; Michaels, C. A.; Stranick, S. J.; Isohashi, T.; Kimura, K. Lett. Org. Chem. 2004, 1, 280. (23) (a) Emerson, E. S.; Conlin, M. A.; Rosenoff, A. E.; Norland, K. S.; Rodriguez, H.; Chin, D.; Bird, G. R. J. Phys. Chem. 1967, 71, 2396. (b) In ref. (a), the salting-out aggregates stained with phosphotungstic acid were used for the TEM measurements. (24) Neumann, B. J. Phys. Chem. B 2001, 105, 8268.

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Figure 1. (a) Absorption spectra of thiacarbocyanine (TCC) dye in aqueous solution at 1.0 × 10-3 mM (green line), 1.0 mM (black line), 4.0 mM (blue curve), and 10 mM (red line). (b) Fluorescence spectra of TCC in aqueous solution at 1.0 mM (black line) and 10 mM (red line). For clarity, the spectrum for [TCC] ) 1.0 mM is arbitrarily magnified.

should be detected directly using polarized-light microscopy. Undoubtedly, applying this technique is significant in the development of a better understanding of structures, morphologies, and formation mechanisms of both the H and J aggregates. In this article, we present investigations of the H and J aggregation behaviors of a thiacarbocyanine dye in solution. We found that the mesoscopic H and J aggregates can be in situ and simultaneously detected by their own colors under the polarized-light microscope. Birefringent properties of the H aggregate having fibrous morphology are determined, so that the molecular arrangements within the fiber are clarified. We also demonstrate the supramolecular dotlike or fibrous morphologies of the J aggregates in solution. Experimental Section Materials. Triethylammonium salt of 3,3′-disulfopropyl-5,5′dichloro-9-methyl thiacarbocyanine (abbreviated as TCC; the chemical structure is shown in the inset in Figure 1b) was purchased from Hayashibara Biochemical Laboratories (Okayama, Japan) and used as received. Pure water was obtained by using an Aquarius GSR-200 (Advantec Co. Ltd.) water distillation system. Sample were prepared as follows: TCC (1.0 × 10-3-10 mM) was dissolved in an aqueous solution under moderate heating, followed by cooling with ice water for 1 h. After keeping the sample solutions for 1-2 days under dark conditions at room temperature, various measurements were conducted.

Mesoscopic H and J Aggregates of TCC Dye Methods. (a) Spectroscopy. Absorption spectra were recorded with a Hitachi U-4100 spectrophotometer, and fluorescence spectra were recorded with a Hitachi F-4500 spectrofluorometer. Rectangular 10- and 1-mm cuvettes were used for the measurements of the solution at [TCC] e 0.01 mM and [TCC] ) 0.1 mM, respectively. For higher concentrated solutions at [TCC] g 1.0 mM, cells with a smaller path length were used. (b) Fluorescence Microscopy. Fluorescence micrographs were obtained with a color CCD camera (HCC-600; Flovel) set on an optical microscope (BX-60; Olympus). A monochromatic excitation beam (∼579 nm) was obtained by passing the light from an Hg lamp (USH102D; Ushio, 100 W) through a mirror cube unit (UMWIY2; Olympus). Fluorescence micrographs under a linearly polarized-light excitation were obtained by using a polarizer (UPO or U-AN; Olympus) mounted in the path of the excitation beam. Fluorescence microspectroscopy was done with a polychromator-multichannel photodetector set (PMA-11; Hamamatsu Photonics) mounted on the optical microscope.21 (c) Polarized-Light Microscopy. In the presence of structural anisotropy, the optical state also displays anisotropic effects (birefringence) in polarized-light observations. Polarized-light microscopy (orthoscopy) was performed with an optical microscope (BXP; Olympus) with crossed polarizers in the absence or presence of a 530-nm retardation plate (U-TP530; Olympus). Adding a compensator to the polarized-light microscope gives us quantitative measurements to determine the relative retardation between the orthogonal wave fronts (ordinary and extraordinary) that are introduced by the specimen birefringence. To obtain relative retardation of the aggregates, a de Se´narmont compensator (U-CSE; Olympus) was placed in the polarized-light microscope under the monochromatic illumination through an interference filter (431F550; Olympus), because the de Se´narmont compensator used is designed at a specific wavelength of 550 nm. The polarized-light micrographs were taken using a Watec WAT-231S color CCD camera. The sample solutions were contained between glass slides and cover slips, and they were sealed with an inert PCTFE (poly(chlorotrifluoroethylene)) grease. (d) Atomic Force Microscopy (AFM). AFM topographic images were recorded using a Nanoscope IIIa (Digital Instruments) operating at the tapping mode with Si microcantilevers (TESP, Digital Instruments) that had a spring constant of 13-100 N m-1. Samples were prepared as follows. After an aliquot of aqueous TCC solution was placed on a mica substrate, the excess of the solution was removed, leaving only a small amount of solution on the substrate. Then the substrate was dried quickly. During the measurements, the drive frequency and the scan rate were ∼270 kHz and 1.0 Hz, respectively.

Results and Discussion Spectroscopic Characterization of J and H Aggregates of TCC Dye in Solution. Figure 1a shows a set of absorption spectra for various TCC concentrations in water: 1.0 × 10-3 mM (green line), 1.0 mM (black line), 4.0 mM (blue line), and 10 mM (red line). The spectrum for the lowest concentration of 1.0 × 10-3 mM comes from the TCC monomer.25 As can be observed in Figure 1a, as the dye concentration is increased to 1.0 mM, the spectrum exhibits a relatively narrow band (475 nm) that is blueshifted in comparison to the monomer peak (548 nm). This blue-shifted spectrum is commonly assigned to H aggregates. Note that the spectral blue-shifts toward the wavelength of 548 nm as a function of the TCC dye concentration were very complicated (see Figure S1 in the Supporting Information), suggesting that the H aggregate observed at 1.0 mM would be a polymeric species. We here focus on this H aggregate. Further increases of concentration still alter the absorption spectra. A sharp and intense J band (636 nm, bandwidth (fwhm) ) ∼360 cm-1), red-shifted to the monomer peak, appeared (25) Noukakis, D.; Van der Auweraer, M.; Toppet, S.; De Schryver, F. J. Phys. Chem. 1995, 99, 11860.

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at above ∼3.0 mM TCC together with the H band. The spectrum at [TCC] ) 4.0 mM shown in Figure 1a indicates the presence of both H and J aggregates in solution. The spectral change arises from an increased amount of J aggregates at the expense of a decreasing H aggregate. At a higher dye concentration (10 mM), the absorption spectra solely exhibited the J band.26 Figure 1b shows the fluorescence spectra of aqueous TCC solutions at concentrations of 1.0 and 10 mM. The Stokes shift for the emission with a high quantum yield observed at 10 mM is quite small (peak wavelength; ∼639 nm), as a characteristic of J aggregates. On the other hand, the emission from the H aggregate (1.0 mM) was very weak, accompanied by a very large Stokes shift. Because the excited energy relaxes rapidly within the excitonic band in the H aggregates down to states with vanishing transition dipole moment, the weak fluorescence with a large Stokes shift is obviously a characteristic of H aggregates.6 Characterization of Mesoscopic Supramolecular J Aggregates by Fluorescence Microscopy. Fluorescence microscopy was conducted to determine the mesostructures of the TCC J aggregates in solution.20,21 Figure 2 shows the concentration dependence of the fluorescence micrographs at concentrations of 1.0-10 mM TCC, representing morphological changes of the TCC J aggregates in solution. At a low dye concentration of 1.0 or 2.0 mM, no characteristic images (or dark images) could be seen, indicating no J aggregates in the solution. At 3.0 mM, dotlike emissive fragments in Brownian motion were observed. We confirmed that the fluorescence spectrum (peak wavelength ) ∼638 nm) obtained under the microscope was quite similar to that in Figure 1b. Considering that the J aggregation occurred at above ∼3.0 mM, the dotlike emissive fragments distributed are the TCC J aggregates. Upon increasing the dye concentration, the morphology of the J aggregate was drastically transformed. Mesoscopic fiber morphology was clearly observed at concentrations of 4.0 and 5.0 mM. The length of the fiber was several tens of micrometers, while the width was very narrowssubmicrometers. This fibrous structure probably comes from anisotropic interactions between TCC molecules in the aggregate, similar to that observed for thiacyanine J aggregates.20-22 With further increase of the TCC dye concentration, the aggregate morphology changed slightly to a linear shape. The direction of the molecular alignment in the J aggregate fiber can be evaluated by examining the fluorescence micrographs of an individual aggregate taken under linearly polarized-light excitation, because excitation efficiency depends on the orientation of the transition moments of the J aggregate which relates to that of the monomer alignment.27 Figure 3a or 3b shows a typical fluorescence micrograph of the J aggregates obtained by excitation with unpolarized or linearly polarized light, respectively. In Figure 3b, the image x-axis corresponds to the direction of the excitation polarization. A bright fluorescence image of the fibers was obtained when the direction of the excitation polarization was parallel to that of the long axis of the fiber, whereas the dark image could be seen when the direction of the excitation polarization (26) At [TCC] ) 10 mM, an absorption shoulder located at 590 nm was also observed along with the intense J band. The shoulder position and the spectral shape did not change within experimental uncertainty. Although the origin of this shoulder is unclear, it would be ascribed either to the vibrationally coupled level of the J band (ref 18) or to Davydov-type splitting of the J band. (27) Marchetti, A. P.; Salzberg, C. D.; Walker, E. I. P. J. Chem. Phys. 1976, 64, 4693.

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Figure 2. Representative fluorescence micrographs of TCC J aggregates in aqueous solution. The concentration was changed between 1.0 and 10 mM.

was perpendicular to that of the long axis (compare Figures 3a and 3b). The observed polarization behaviors of the J aggregates indicate that the long axis of each of the TC molecules was approximately aligned parallel to that of the fiber. In Situ Imaging of Mesoscopic H and J Aggregates by Polarized-Light Microscopy. Polarized-light microscopy is a useful technique for detecting regular molecular alignments in the aggregate having strong birefringence, since it can distinguish between isotropic and anisotropic regions.28 In the course of the birefringence observation of the TCC J aggregates using polarized-light microscopy, we succeeded in visualizing the mesoscopic morphologies of the J and H aggregates simultaneously. Figure 4 shows typical polarized-light microscope images with crossed polarizers. Note that no retardation plates or compensators are inserted into the microscope. At [TCC]

) 1.0 mM, the bright image with a pale-blue color clearly exhibited fibrous morphology with strong birefringence. The size of the fiber was > ∼80 µm in length and submicrometers in width. At 2.0 mM, the number and the length of the fibers increased. Because the absorption spectra of TCC showed the presence of pure H aggregates at these concentrations (1.0 and 2.0 mM), the observed fibers with strong birefringence should be the H aggregates distributed in solution. We could not see the H aggregates with fluorescence microscopy due to a small fluorescence quantum yield of the aggregate. With a further increase of the dye concentration, the H and J aggregates that coexisted could be clearly distinguished. At 3.0 mM, an approximate threshold concentration of J aggregation, reddish bright dots appeared along with elongated bluish fibers of H aggregates (Figure 4c). Compared to the fluorescence microscope image (Figure 2c), the reddish dots observed with polarized-light microscopy correspond to the J aggregate of TCC. At the range of 4.0-10 mM TCC, the morphology of the J aggregate changed from a fragmented dot into a fiber, consistent with the morphological changes observed with fluorescence microscopy. Under the observations of densely dispersed J aggregates in solution (Figures 4d-4f), the micrographs exhibited vague birefringent textures due to a stacking of the fibers. On the other hand, the bluish fibers decreased in number with an increase in the TCC concentration, indicating a decrease of H aggregates. Coexistence of mesoscopic H and J aggregates in solution would be a rare phenomenon in the absence of any templates.29 The H and J aggregate morphologies could be distinguished by their own colors under the polarized-light microscope. The observed color differences can be explained via two mechanisms as described here. The first mechanism involves the differences in the intrinsic interference colors between the H and J aggregates. Under polarized-light illumination, a birefringent material produces two individual out-of-phase light components that are each perpendicularly polarized. The faster component emerges first from the sample relative to the slower one with an optical path difference (or retardation). The

(28) Hamley, I. W. Introduction to Soft Matter - Polymers, Colloids, Amphiphiles and Liquid Crystals; John Wiley & Sons: Chichester, 2000.

(29) Chowdhury, A.; Wachsmann-Hogiu, S.; Bangal, P. R.; Raheem, I.; Peteanu, L. A. J. Phys. Chem. B 2001, 105, 12196.

Figure 3. Fluorescence micrographs of fibrous J aggregates obtained by using (a) unpolarized and (b) linearly polarizedlight excitation. These images were taken at the same view area. The excitation polarization in image (b) is oriented along the image x-axis. According to the fibers indicated by arrows in image (a), a bright image could be obtained when the direction of the excitation polarization was parallel to that of the long axis of fibers.

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Figure 4. Representative polarized-light micrographs with crossed polarizers. Species observed in pale-blue or red color correspond to the H and J aggregates of TCC, respectively. The concentration was changed between 1.0 and 10 mM.

analyzer in the microscope recombines two components traveling in the same direction and vibrating in the same plane, producing an intrinsic interference color of light. Therefore, the interference color strongly depends on the retardation, as a function of the sample thickness and the degree of birefringence. If retardation is different between the H and J aggregates of TCC, the observable interference colors also differ from each other. The second mechanism comes from the differences in the absorption color between the H and J aggregates. These aggregates intensely absorb different wavelength regions of light; therefore, white light illumination under the polarized-light microscope can provide different transmissions of colors. It is quite reasonable that the observable colors should be the superposition of absorption colors and the intrinsic interference colors. Quantitatively, the intensity of observable light through interference is proportional to the square of the amplitude of two recombined waves (that is, proportional to sin2(π‚R/λ0), where R and λ0 are the retardation and the wavelength of light, respectively).30 At R < ∼150 nm, the value of sin2(π‚R/λ0) monotonically decreases with increasing λ0 in the visible region (∼350900 nm), whereas that oscillates from 0 to 1 at R > ∼150 nm. Therefore, it is conceivable that a pale-blue color of the H aggregate is caused by a relatively small retardation of the aggregate (∼150 nm), and the resulting lack of blue-light region would produce a characteristic interference color of the aggregates. Birefringent Properties of the Fibrous H Aggregates. When the aggregate fiber is rotated with respect to the polarizers, the intensity of the image colors varies cyclically, from zero (extinction) up to a maximum after (30) Kato, T. J. Geol. Soc. Jpn. 2001, 107, 64.

45°, and back down to zero after 90° rotation. Note that the fibrous H aggregate exhibited a straight extinction; namely, the image disappeared when the long axis of the fiber was parallel to the direction of polarization of the analyzer (extinction direction, the image y-axis in Figure 5). This property indicates a uniaxial crystalline nature of the aggregate. Figure 5a shows a typical polarized-light micrograph of the fibrous H aggregate ([TCC] ) 1.0 mM) at a northwestsoutheast orientation (maximum level of birefringence) with crossed polarizers. Under this condition, the H aggregate shows pale-blue color. The observed fiber was ∼100 µm long, and the tips of the fiber were branched, suggesting coalescence or bundling of thin fibers. When a 530-nm retardation plate is placed into the polarizedlight microscope, namely, a known optical path difference of 530 nm is added to the entire field, the interference colors were transformedswe can see an isotropic region as magenta. Figure 5b shows the image of the same H aggregate taken in the presence of the 530-nm retardation plate. Despite a poor-contrast image due to small retardation of the aggregate, the fiber of interest showed bluish. Analyzing the image colors shown in Figure 5b, we can evaluate the degree of birefringence (∆n) of the H aggregates. Here, ∆n ) nL - nS, where nL or nS represents the refractive index along the long or short axis of the H aggregate fiber, respectively. Although a quantitative ∆n value cannot be obtained because of the intense absorption of the H band, we can determine the sign of ∆n by examining the interference colors of the specimen that depend on the azimuthal angle from the extinction direction. If the specimen slow axis (namely, the axis in the high refractive index) is parallel to the slow axis of the retardation plate (northeast-southwest direction), the additive retardation effects result in higher-order interference colors (blue color as deduced on the basis of the interference color chart30) superimposed on an isotropic magenta background. Therefore, Figure 5b indicates that the short axis of the fibrous H aggregate corresponds to the slow axis. The result proves negative birefringence of

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Figure 5. Polarized-light micrographs of the single fibrous H aggregate ([TCC] ) 1.0 mM) at northwest-southeast orientation (maximum level of birefringence) with crossed polarizers (a) in the absence of any retardation plate or compensator, (b) in the presence of a 530-nm retardation plate, (c) in the presence of de Se´narmont compensator at the analyzer angle of 0°, and (d) in the presence of the de Se´narmont compensator at the analyzer angle of 11°.

the H aggregates: nL < nS. Since the refractive index is positively correlated with electronic polarizability via the Lorenz-Lorentz relation,4 a larger refractive index is caused by larger electronic polarization through delocalized π-electron systems in the TCC molecule. The negative birefringence for the fiber structure suggests that TCC molecules are likely to orient with respect to the shortaxis direction of the fiber. A de Se´narmont compensator placed into the microscope can be used for quantitative retardation measurements over the range of approximately 550 nm when observed with a monochromatic 550-nm line. By using this compensator, we can estimate the relative retardation of the H aggregate of TCC. Note that the sign of birefringence and orientation of the specimen slow axis must be known before the measurements. At the northwest-southeast orientation for the negative birefringent H aggregates, we can see a bright-green image of the fiber on the dark background (Figure 5c). Compensation is determined by rotating the analyzer from its crossed position through a measured angle (θ, in degrees) until the intensity of the fiber is minimized (extinction; shown in Figure 5d). The background is brightened from black to green as the analyzer is rotated to obtain extinction. In the case of this H aggregate fiber, θ ) ∼11° was determined. Under a monochromatic light illumination of 550 nm, the relative retardation (Γ) can be calculated by the following equation: Γ ) (550 × θ)/180. In this way, we found the Γ value to be ∼34 nm. Topographic Study of the Fibrous H Aggregates. To further clarify the nanoscopic structure of the fibrous H aggregates, AFM was carried out. The samples were prepared in which a small drop of solution (1.0 mM) was quickly dried on a mica substrate. Figure 6 shows a typical

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Figure 6. Typical AFM image and a cross-sectional profile of a fibrous H aggregate.

AFM image of a fibrous H aggregate along with a crosssectional profile. The fibers were ∼30-100 nm wide and ∼10-15 nm high. The tip of the fibers was branched, indicating that the thick fiber was a bundle of primary thin fibers. The result can be confirmed by the polarizedlight micrographs shown in Figure 5. Although the height of the fibers was modified by the removal of water through a drying process for the AFM observation, the thickness of the primary fiber of the solution H aggregates would be in the range of ∼15-30 nm, which corresponds to the aggregation number of 105-106 when the fiber length is ∼100 µm.31 For the H aggregate fiber, Γ was estimated to be ∼34 nm in the preceding section. Assuming that the thickness of the H aggregate fiber, d, (in this case, the fiber means a bundle of primary fibers) is on the order of 100 nm, we can calculate the degree of birefringence, namely -∆n ) Γ/d, to be ∼-0.3. Such a very large birefringence, similar to that for birefringent aromatic liquid crystals,32 would arise from anisotropic well-ordered molecular alignments within the aggregates. It should be noted that a mesoscopic rodlike morphology of H aggregates has been observed by using ex situ TEM for a certain thiacarbocyanine dye, whose chemical structure is similar to that of the dye we used but different in the side chain groups.23 Although the morphology of the H aggregates resembled that of our TCC dye, their dye did not produce solution-phase J aggregates, probably as a result of the difference in the side chains. The results imply that not only the chromophore but also the side(31) The TCC molecule possesses a π-electron chromophore of ∼1.6 × ∼0.5 nm and sulfopropyl groups of ∼0.6 nm. (32) Kelker, H.; Scheurle, B. Angew. Chem. 1963, 81, 903.

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chain structures greatly influence the molecular arrangements and the relevant mesoscopic morphology of the supramolecular aggregates. Mesoscopic Reassembly from H to J Aggregates in Solution. In the aggregation of TCC, we revealed that mesoscopic supramolecular H aggregates transformed into J aggregates with similar morphology upon increasing the dye concentration. On the other hand, studies on the PIC dye showed that the H aggregates, whose aggregation number determined by the static light scattering was on the order of ∼30,24 reassembled further into supramolecular J aggregates with the large aggregation number of ∼3000 upon increasing the dye concentration.17 Remarkable differences are present between the TCC and PIC systems as follows: (i) an enhanced growth in aggregate size (or length) was not observed upon J aggregation of TCC, (ii) the aggregation number of TCC molecules was 2-3 orders of magnitude larger than that of PIC. In both cases, however, the results on the aggregate transformation as a function of the dye concentration4 indicate that J aggregates should have a more compact arrangement of constituent molecules compared to the H aggregate. This consideration is supported by the highpressure experiments on concentrated PIC solutions, which shows that the molecular density of the J aggregate is higher than that of the H aggregate.33 Molecular reassembly in the aggregate at high dye concentrations is probably induced according to energetical and/or entropical requirements,33 and the details of molecular packing demand further investigations. Conclusions We have characterized mesoscopic structures of both supramolecular H and J aggregates of 3,3′-disulfopropyl(33) Neumann, B.; Pollmann, P. Phys. Chem. Chem. Phys. 2000, 2, 4784.

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5,5′-dichloro-9-methyl thiacarbocyanine (TCC) dye in aqueous solutions (1.0-10 mM). In situ distinction between the H and J aggregates can be made by their own colors using polarized-light microscopy. The TCC dye formed pure H aggregates in aqueous solution at 1.0 or 2.0 mM. The H aggregate showed mesoscopic elongated fiber morphology with negative birefringence, indicating that the refractive index along the fiber short axis was higher than that of the long axis. In the H aggregate, physical aggregation number is estimated to be on the order of ∼105-106; therefore, π-electron chromophores are likely to orient along the short axis of the fibers. The degree of birefringence of the H aggregate was also determined quantitatively. At [TCC] ) 3.0 mM, a small dot morphology of the J aggregates appeared together with fibrous H aggregates. Upon further increasing the dye concentration (g4.0 mM), the dotlike J aggregates changed into mesoscopic fibers, similar to those of the H aggregates. In contrast, fluorescence microscopy under linearly polarizedlight excitation revealed that the constituent molecules within the fibrous J aggregates are aligned along the long axis of the fibers, implying positive birefringence of J aggregates.34 Acknowledgment. H.Y. is indebted to financial support from the Hyogo Science and Technology Association. Supporting Information Available: Absorption spectra of TCC dye in aqueous solution at 0.01, 0.1, and 1.0 mM. This material is available free of charge via the Internet at http://pubs.acs.org. LA0479004 (34) It is worth noting that positive birefringence of the fibrous J aggregates of TCC was also verified by examining the interference colors of the aggregates under the polarized-light microscope in the presence of the 530-nm retardation plate.