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Spatial and Spectral Heterogeneity in Fluorescence from Monolayers of 4-(4-(Dihexadecylamino)styryl)N-methylpyridinium Iodide Amy L. Lusk and Paul W. Bohn* Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801 Received April 24, 2000. In Final Form: September 1, 2000 Far-field fluorescence of Langmuir-Blodgett (LB) films of the hemicyanine dye, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide, I, is spectrally heterogeneous, displaying bands assigned to monomer and various aggregated species in both excitation and emission. LB films of I visualized by epifluorescence microscopy were also observed to be spatially heterogeneous. Under the preparation conditions used here the morphology consists of coexisting liquid expanded (LE) and liquid condensed (LC) phases. LC domains display distinct spatial structure. They are ca. 5-20 µm in lateral size, display a multilobed shape, and are surrounded by LE phase. The characteristic aggregation behavior of these stilbazolium-based hemicyanines allowed their electronic spectra to be used to provide the contrast in fluorescence microscopy of LB films of I. Emission images thus reveal the aggregation-induced electronic structure of the molecules composing the domains. The degree of internal structure of the LC domains was observed to vary with excitation wavelength with the richest internal structure being observed with UV excitation of the fully developed H-aggregate. Correlation of film and solution fluorescence spectra suggest that the LC domains are composed of dimers, trimers, and larger n-mers up to the fully extended aggregate, while the LE phase is predominantly populated by monomers with some fully extended aggregates.
Introduction Hemicyanines, which exhibit large molecular hyperpolarizabilities (∼2 × 10-27 esu) and are readily incorporated in amphiphilic molecular structures, are of potential interest for molecular optics, optoelectronics, and telecommunications.1-6 However, fabrication of films with the appropriate collective nonlinear optical properties has proven difficult. A common theme among potential explanations is the tendency for aggregation which results in microheterogeneity within Langmuir-Blodgett (LB) films of hemicyanines.7-11 Microheterogeneity in assemblies of dye molecules is often associated with anomalous spectroscopic features and is ascribed to intermolecular excited-state interactions. The lateral interaction of chromophore units results in spatially extended electronic states, with aggregationnumber-dependent energies blue-shifted from that of the monomer S1 state. Further, excitonic energy transport dominates the radiative dynamics.12-15 The extended * To whom correspondence should be addressed, bohn@scs. uiuc.edu. (1) Liu, X.; Liu, L.; Chen, Z.; Lu, X.; Zheng, J.; Wang, W. Thin Solid Films 1992, 219, 221-225. (2) Kajikawa, K.; Anzai, T.; Shirota, K.; Takezoe, H.; Fukuda, A. Thin Solid Films 1992, 210, 699. (3) Kajikawa, K.; Shirota, K.; Takezoe, H.; Fukuda, A. Jpn. J. Appl. Phys. 1990, 29, 913. (4) Shirota, K.; Kajikawa, K.; Takezoe, H.; Fukuda, A. Jpn. J. Appl. Phys. 1990, 29, 750. (5) Gao, J.; Darling, G. J. Am. Chem. Soc. 1992, 114, 3997-3998. (6) Marowsky, G.; Chi, L.; Mobius, D.; Steinhoff, R.; Shen, Y.; Dorsch, D.; Rieger, B. Chem. Phys. Lett. 1988, 147, 420-424. (7) Lupo, D.; Prass, W.; Scheunemann, U.; Laschewsky, A.; Ringsdorf, H.; Ledoux, I. J. Opt. Soc. Am. B 1988, 5, 300. (8) Ashwell, G.; Hargreaves, R.; Baldwin, C.; Bahra, G.; Brown, C. Nature 1992, 357, 393. (9) Carpenter, M.; Willand, C.; Penner, T.; Williams, D.; Mukamel, S. J. Phys. Chem. 1992, 96, 2801. (10) Marowsky, G.; Steinhoff, R. Opt. Lett. 1988, 13, 707-709. (11) Cnossen, G.; Drabe, K.; Wiersma, D. J. Chem. Phys. 1992, 97, 4512.
dipole model16 ascribes the blue-shifted spectral features to perturbation of the isolated molecule energy levels by lateral intermolecular interactions. In this model the molecular packing determines the extent and sign of spectral displacement with the two limiting cases involving evolution of the S1 molecular state to aggregate states at higher (H aggregate, k > 0) or lower (J aggregate, k ) 0) energy. Because the dispersions of the H- and J-aggregates are of opposite sign, the low energy state of the J aggregate is at k ) 0, resulting in superradiant behavior, while the H aggregate decays only through nonradiative pathways.17-19 The hemicyanine dye used in this study, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide, I, forms H-aggregates both in LB films and in solution. Previous studies from this laboratory utilized the related hemicyanine dye II. The presence of the negatively charged propylsulfonate group has a major impact on the packing of this dye compared to dye I (R ) CH3). Most strikingly the aggregation behavior is very different for the two hemicyanines, with the propylsulfonate pyridinium species II aggregating strongly and spontaneously, while the methyl pyridinium dye I forms the aggregate state much more slowly, so that it can be prepared with a heterogeneous distribution of aggregation states.20 Our earlier (12) Hall, R. A.; Thistlethwaite, P. J.; Grieser, F.; Kimizuka, N.; Kunitake, T. J. Phys. Chem. 1993, 97, 11974-11978. (13) Kajikawa, K.; Shirota, K.; Takezoe, H.; Fukuda, A. Jpn. J. Appl. Phys. 1991, 30, 362-365. (14) Furman, I.; Geiger, H. C.; Whitten, D. G.; Penner, T. L.; Ulman, A. Langmuir 1994, 10, 837-843. (15) Song, X.; Geiger, C.; Leinhos, U.; Perlstein, J.; Whitten, D. G. J. Am. Chem. Soc. 1994, 116, 10340-10341. (16) Czikkely, V.; Forsterling, H.; Kuhn, H. Chem. Phys. Lett. 1970, 6, 207. (17) Muenter, A.; Brumbaugh, D.; Apolito, J.; Horn, L.; Spano, F.; Mukamel, S. J. Phys. Chem. 1992, 96, 2783. (18) Spano, F.; Mukamel, S. J. Luminesc. 1990, 45, 412. (19) Dubovsky, O.; Mukamel, S. J. Chem. Phys. 1992, 96, 9201. (20) Song, Q.; Xu, Z.; Lu, W.; Bohn, P. W. Colloids Surf. A 1994, 93, 73-78.
10.1021/la000599g CCC: $19.00 © 2000 American Chemical Society Published on Web 10/11/2000
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studies demonstrated that a particularly important aggregate decay pathway involves energy transfer to monomer species,21 this fact having been used to estimate the upper limit on H-aggregate size for hemicyanine II.22 The much richer array of structural possibilities which occur in monolayers of I is associated with a more diverse set of relaxation phenomena and make this dye a good choice for studying the relationship of spatial and spectroscopic heterogeneity.
Figure 1. Absorption spectra of I in solution (s) and in an LB monolayer (- - -). The spectra have been normalized to equivalent absorbance for the strongest band in each spectrum.
Spatially integrated measurements of ensemble average fluorescence and absorbance are commonly used to study monolayers containing dye molecules. However spatially resolved imaging is critical, if the physical origins of spectroscopic heterogeneity observed in these films are to be understood. Spatial surveys of phase behavior in lipid monolayers and bilayers are commonly performed via epifluorescence microscopy,23-25 by incorporating a fluorescent membrane lipid analogue into the lipid layer at low mole fraction and using it to report on the behavior of the dominant membrane constituent. Such studies reveal liquid condensed (LC) domains of micrometer characteristic dimensions surrounded by liquid expanded (LE) domains, with the size and shape of the domains changing with preparation conditions. Although the same general behavior might be expected for fluorescent lipid analogues such as I, the literature contains little evidence of this behavior.26 Because the excitation band for aggregates of I is shifted 130 nm blue relative to the monomer band, judiciously designed epifluorescence experiments can reveal both the phase behavior and the predominant composition (aggregate/monomer) of those phases. Such studies should illuminate details of aggregate formation and eventually facilitate using these species in molecular devices, by elucidating the structural basis of microheterogeneity in hemicyanine LB films. This Letter correlates images of spectroscopic microheterogeneity in LB films of I with the corresponding far-field fluorescence and absorption data to relate physical structure to spectroscopic behavior.
H2O2/H2SO4) followed by rinsing with copious amounts of deionized H2O. Monolayers were fabricated using a KSV Instruments (model 5000) Langmuir-Blodgett trough. Substrates were immersed into the subphase prior to spreading amphiphile I onto the subphase. A total of 5 × 1016 ((3 × 1016) molecules were spread from a solution of I in CHCl3 (Burdick & Jackson LC grade) onto a deionized (18 MΩ cm) water subphase (Millipore Corp., model Milli-Q UV plus) at 300 K. One hour was allowed for solvent evaporation and equilibration. Monolayers were typically compressed at a rate of 1.0 ( 0.5 Å2/(molecule/min) to 30 mN/m. LB monolayers were transferred in Y-configuration by vertical deposition. Monolayer Characterization. Absorbance measurements on transferred films were obtained using a commercial doublebeam UV-visible spectrometer (Cary-3, Varian Instruments) at normal incidence. Fluorescence measurements were obtained on two different systems. The first was a commercial dual monochromator system (Spex, model DM1B) with a thermoelectrically cooled photomultiplier tube. Solution spectra were taken in right angle configuration, while transferred film spectra were acquired in a front face configuration with the emission collected at 23° from the excitation axis. The second utilized the ultraviolet line (363 nm) of an argon ion laser as a source and a Spex single monochromator and liquid nitrogen cooled CCD as detector. The geometrical arrangement placed the excitation and collection axes perpendicular to each other with the sample at approximately 40° with respect to the collection axis. Epifluorescence microscopy was performed on an inverted microscope (Zeiss Axiovert 100) equipped with a 150-W Hg lamp, a color camera (Sony Medical Instruments, model DCX 9000), and a thermoelectrically cooled black and white camera (Hamamatsu). Several filter sets (Chroma Inc.) were employed including the following excitation/emission pairs: 360(40)/400LP, 480(30)/535(40), 545(35)/610(75). In each set the center wavelength (bandpass) pair are designated, except the emission filter for the UV set was a 400 nm long wavelength pass filter.
Experimental Section
Results and Discussion
Sample Preparation. The hemicyanine dye, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide, was obtained from Molecular Probes and used without further purification. Fused silica substrates (Heraeus-Amersil) were prepared by immersion in freshly prepared piranha cleaning solution (1:4
As shown in Figure 1, I in solution exhibits two absorbance peaks: one due to the lowest-lying π-π* transition of the monomer at 491 nm; the other at 275 nm is a transition isolated on the pyridinium and phenyl rings. It is well-known that hemicyanines exhibit an extraordinary propensity for aggregation.14,21,26-28 This is illustrated in the absorption spectrum of a transferred monolayer by the appearance of a blue-shifted peak near 350 nm. In addition there is a small blue shift of the free monomer peak to 468 nm. Interestingly solution fluores-
(21) Evans, C. E.; Bohn, P. W. J. Am. Chem. Soc. 1993, 115, 33063311. (22) Song, Q.; Bohn, P. W.; Blanchard, G. J. J. Phys. Chem. B 1997, 101, 8865-8873. (23) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171. (24) Chi, L. F.; Anders, M.; Fuchs, R.; Johnston, R.; Ringsdorf, H. Science 1993, 259, 213-216. (25) Mohwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (26) Fang, J. Y.; Uphaus, R. A.; Stroeve, P. Thin Solid Films 1994, 243, 450-454.
(27) Song, Q.; Evans, C. E.; Bohn, P. W. J. Phys. Chem. 1993, 97, 13736-13741. (28) Befort, O.; Mobius, D. Thin Solid Films 1994, 243, 553-558.
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Figure 2. Fluorescence excitation (left) and emission (right) spectra for I in CHCl3 as a function of concentration: (s) 8.77 µM; (‚ ‚ ‚) 43.9 µM; (- - -) 110 µM; (- - -) 219 µM. The inset shows the shift of the emission λmax with concentration.
cence excitation spectra of I as a function of concentration (Figure 2) also show a strong blue shift (and decrease in intensity) with increasing concentration. Solution emission spectra exhibit a strong band centered near 590 nm, which shifts to longer wavelengths with increasing concentration; cf. Figure 2 inset. Concomitant blue-shifting excitation and red-shifting emission spectra are a characteristic signature of H-aggregate formation, indicating that formation of aggregated species proceeds even in solution. Fluorescence excitation spectra of the LB film (Figure 3) closely resemble film absorption with more definition to the shoulder at 431 nm, while film emission spectra excited at the aggregate wavelength (363 nm) exhibit a broad band centered at λmax ) 517 nm but with significant emission extending out as far as 700 nm. Excitation at the 431 nm shoulder of the excitation spectrum results in an emission band at 495 nm, while excitation at 460 nm shifts the emission maximum to 505 nm, both bands being ca. 80 nm wide. The observation that both solution and LB film fluorescence exhibit structured excitation spectra suggests that multiple species with distinct electronic character are present. Alternatively the structure could originate from vibronic bands. This latter possibility is unlikely, however. The resonance Raman spectrum of dye I displays strong bands at 1575, 1176, and 880 cm-1, all of which are integral to the aromatic π-system.29 Using the band maxima in the solution excitation spectra from Figure 2 and the known vibrational bands can produce some of the features in the excitation spectra, but doing so requires the use of different vibrational bands at each λmax. Additionally, the appearance of a blue-shifted excitation spectrum together with a red-shifted emission spectrum, as seen in solution, strongly suggests that these distinct (29) Cao, X.; McHale, J. L. J. Chem. Phys. 1998, 109, 1901-1911.
electronic states result from aggregate formation.30 The interaction integral, J12, can be calculated in the extended dipole approximation and used to predict the shift in S0S1 transition energy associated with the interaction of neighboring molecules.31 It is given by
J12 )
(
)
2 1 1 1 1 + - D a1 a2 a3 a4
(1)
where is the partial charge (2.12 × 10-10 esu) associated with the optical transition, D is the dielectric constant ∼2.5, and the an terms are the separation of partial charges on neighboring transition dipoles. The shift of the aggregate transition, Eaggr, relative to that of the monomer, Emono, is determined here by simply summing nearestneighbor couplings.32 Taking the position of the monomer absorption band in solution at 491 nm as the unperturbed state, and using known structural parameters, an, the shifts due to interaction of a monomer with one to six molecules (n ) 2-7) are summarized in Table 1. The correspondence of the calculated absorption energies for n-mers, 2 e n e 4, with the observed spectral shifts both in solution and in LB monolayers is striking, suggesting the presence of n-mers of varying size in both solution and LB films. While the presence of n-mers with n e 6 is indicated by the spectroscopic data, little can be said about the existence of larger aggregates, because the simple model used here only accounts for nearest-neighbor interactions. Aggregate structures with n > 7 would all appear at the H-aggregate wavelength in this simple model. Prior work from this laboratory on hemicyanine (30) Kuhn, H.; Czikkely, V.; Fo¨sterling, H. Chem. Phys. Lett. 1970, 6, 11. (31) Evans, C. E.; Song, Q.; Bohn, P. W. J. Colloid Interface Sci. 1994, 166, 95-101. (32) Bohn, P. W. Annu. Rev. Phys. Chem. 1993, 44, 37-60.
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Figure 3. Fluorescence excitation spectrum (λmax ∼ 344 nm) and emission spectra excited at different n-mer wavelengths for an LB monolayer of I. Emission spectra were excited at: (s) 344 nm; (‚ ‚ ‚) 433 nm; (- - -) 456 nm. Table 1. Calculated and Observeda Values of n-mer Absorption and Fluorescence Excitation Band Positions (nm) calculatedb Emono
491
Emono + 1(J12) Emono + 2(J12) Emono + 3(J12) Emono + 4(J12) Emono + 5(J12) Emono + 6(J12)
459 431 406 384 364 346
8.77 µM
43.9 µM
494 484 470 450 435
492 481 468 452 436
110 µM
219 µM
LB film
453 432 411
455 432 412
455 432
344
a
The wavelength of maximum emission, λmax, is indicated in boldface for each sample condition. b The origin of the calculated series is taken from λmax of the solution absorption at the lowest concentration.
II, however, showed an upper limit to the coherence length of the aggregate of N e 50.22 The widely used pincushion model17-19,33 for H aggregate formation dictates a nonradiative decay of the aggregate species, because decay occurs from a k * 0 state. Previously the nonradiative decay of the zwitterionic hemicyanine II has been interpreted within this approximation by positing two primary decay mechanisms; exciton hopping followed by Fo¨rster energy transfer to the monomer population, or direct coupling of the exciton energy to the phonon bath.21,22 However, hemicyanine I undoubtedly packs differently than II as evidenced by the more varied solution excitation spectra and the fact that aggregates form much more slowly in systems of I than in II. Thus it is not surprising that the present data indicate a rich array of decay possibilities for aggregates of I, including, in addition to the above possibilities, energy transfer to specific n-mers (33) Hochstrasser, R. M.; Kasha, M. Photochem. Photobiol. 1964, 3, 317-331.
and direct aggregate emission. As shown in Figure 3 excitation of the LB film at wavelengths corresponding to any particular n-mer in the range 2 e n e 4 produces emission lying within the broad emission envelope derived from excitation of the H-aggregate at 363 nm, thus indicating energy transfer. Figure 4 presents a series of fluorescent images obtained under varying excitation and emission conditions. Much like studies on monolayers of lipids, the domains in these films are attributed to the LE and LC phases. Despite the fact that the films are chemically homogeneous, the phase boundaries can be imaged without addition of a diluent, because they are spectroscopically heterogeneoussthe contrast being provided by the varying aggregation states present in different spatial regions. At 40× magnification the resolution limit is ca. 1 µmsnot sufficient to see individual aggregates whose electronic structure has an upper limit of 50 molecules.22 However, the predominant species in each phase can be determined, and it is clear that the island features, assigned to the LC phase, have definite internal structure. Under ultraviolet (360 nm) irradiation the H-aggregates are excited and can undergo Fo¨rster energy transfer to either n-mer or monomer species. Panels A and D of Figure 4 show gray scale and color emission images, respectively, in the range λem > 400 nm when excited at 360 nm. Figure 4A shows three distinct gradations in gray scale intensity, from the center to the edge of the LC domains, presumably corresponding to regions with varying aggregate excitation efficiency, energy transfer efficiency, or a combination of the two. It is interesting that while the size and shape of islands varies from island-to-island, the form of the internal structure is quite similar among islands, with the center and edges darker than an intermediate light region. In addition to the structured spatial emission from LC domains there are a number small bright regions which are scattered and encountered in both LC and LE domains.
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Figure 4. Fluorescence microscopy images for an LB monolayer of I transferred at π ) 30 mN/m collected with the following filter sets: (A) 360(40)/400LP; (B) 480(30)/535(40); (C) 545(35)/610(75); (D) 360(40)/400LP. Images A and C display the same physical region of the film, while images B and D display different regions.
These regions are a characteristic feature of LB films of I prepared under long-time and large transfer pressure conditions. Their origin and temporal evolution will be the subject of a forthcoming publication.34 Figure 4D, acquired under the same conditions as the image in Figure 4A, shows that the wavelength integrated emission is spatially segregated, with distinct regions of green and orange emission, consistent with the spatially integrated fluorescence emission data in Figure 3 which shows an emission band encompassing both green and orange wavelengths. Figure 4B presents fluorescence excited at 485 nm and collected at 535 nm. Under these conditions the center of the LC islands is dark, and only an intermediate area of the LC domain emits strongly in the green region. Color images (not shown) do, however, indicate that the surrounding LE phase fluoresces green, though not as intensely. It is striking that when the excitation is carried out on the red edge of the excitation profile, Figure 4C, the LC islands are much more uniform in their emission intensity. The color image in Figure 4D shows that the mole fraction of monomer (green emission) is greater in the LE domains than in the LC domains, while emission from the LC domains is dominated by orange emission. Given (34) Lusk, A.; Bohn, P. Submitted for publication in J. Phys. Chem. B.
the monotonic red shift of the emission λmax with increasing solution concentration of I (cf. Figure 2), it is reasonable to posit the existence of small aggregates, or n-mers with emission energies below that of the monomer. Correlation of film and solution fluorescence spectra indicates that these n-mers contribute more strongly to the emission in the LC regions than in the LE regions. This suggests that the LC domains are composed of dimers, trimers, and larger n-mers up to the fully extended aggregate, while the LE phase is predominantly populated by monomers with some fully extended aggregates. However, because the film is excited at the 360 nm absorption of the fully extended aggregate, the aggregate must exist in both phases in order that emission from both phases can be observed simultaneously. Under extended irradiation (1530 s) orange emission from the LC phase shifts to yellow, then green, and then disappears, likewise the green emission of the LE phase fades. This photobleaching is largely irreversible and presumably results from photodimerization to a nonfluorescent squarine species. Conclusion The strong propensity for the hemicyanine I to form electronically aggregated species produces a rich variety of spatial structures and spectroscopic behavior. The resulting heterogeneity occurs on length scales spanning the range from the size of an individual n-mer (ca.1 nm) to the scale of the internal structure of the LC domains
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visible in Figure 4. Previous work in our laboratory and others devoted to characterizing molecular assemblies of hemicyanines has looked solely at spatially integrated behavior. The current work reveals rich structure in the spectroscopic morphology on the micrometer length scale, which undoubtedly underlies the seemingly anomalous optical behavior of assemblies of hemicyanines. Since spectroscopic behavior is closely linked to the morphologies present in a given sample, and a variety of global structures can possess nearly the same energy, it is perhaps not surprising that small differences in preparation conditions can lead to large differences in the observed spatially
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integrated behavior. The spatially resolved behavior explored here demonstrates the rich variety of microstructure which exists and gives rise to varying spatially integrated emission. Acknowledgment. This work was supported by the National Science Foundation throughGrant CHE 9910236 and by the Department of Energy through Grant DE FG0288ER13949. LA000599G
