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Bidimensional Distribution of a Cyanine Dye in Langmuir-Blodgett (LB) Monolayers Studied by Time-Resolved and Spatially Resolved Fluorescence H. Laguitton-Pasquier, M. Van der Auweraer,* and F. C. De Schryver Laboratory for Molecular Dynamics and Spectroscopy, Chemistry Department, K U Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium Received July 15, 1997. In Final Form: April 8, 1998 The excitation energy transfer from monomers to dimers of N,N′-dioctadecyl thiacyanine incorporated in Langmuir-Blodgett (LB) films of Cd-arachidate or dipalmitoyl phosphatidic acid was studied as a function of the dye concentration, ranging from 0.5 to 6 mol %, to obtain insight in the two-dimensional distribution of the dye. The details of the energy transfer were determined by the global analysis of picosecond time-resolved fluorescence decays recorded on a single mixed monolayer. Those studies were correlated with scanning confocal fluorescence microscopy measurements. From the spectral decomposition of the absorption spectra, the fraction of molecules present as dimers or aggregates amounts to 0.2 and is constant over the dye concentration range studied. The spatially unresolved data are supported by the confocal experiments which show that the dye distribution in the LB films studied is inhomogeneous and suggests the formation of two separate phases: strongly fluorescing regions of high dye concentration, containing a mixture of monomers and dimers participating in the energy transfer process, are surrounded by regions of low fluorescence intensity and of small dye concentration containing almost exclusively dimers. Although in the Cd-arachidate matrix the fraction of dimers not participating in the energy transfer process amounts to 0.135 ( 0.015 over the concentration range studied, this amount can be considered negligible in the dipalmitoyl phosphatidic acid matrix.
Introduction Many specific properties as well as potential applications of Langmuir-Blodgett (LB) films are related to their molecular order1-4 which can differ from that in bulk materials.5,6 Since the pioneering work by Kuhn,7 his proposal for “molecular engineering”8 and the studies by Roberts on the applications of LB films in electronics,9 the use of LB films was clearly established as an important technique to investigate energy or charge transfer on a molecular scale.1,2,10-13 The occurrence of those processes will depend on the spatial structure in the plane of the monolayers, so the characterization of the arrangement and the spatial distribution of the monolayer constituents appears to be an essential step in the understanding of the photophysical * To whom correspondence should be addressed. (1) Kuhn, H.; Mo¨bius, D.; Bucher H. In Techniques of Chemistry; Weisberger, A., Rossiter, B. W., Eds.; Physical Methods of Chemistry Series; Wiley: New York, 1972; Vol. 1, Part IIIB. (2) Kuhn, H.; Mo¨bius, D. Investigations of Surfaces and Interfaces Part B, 2 ed.; Rossiter, B. W., Ba¨tzold, R. C. Eds.; Physical Methods of Chemistry Series; Wiley: New York, 1993; Vol. IXB. (3) Langmuir-Blodgett Films; Roberts, G., Ed.; Plenum: New York, 1990. (4) Gaines, J. L. Insoluble Monolayers at Liquid-Gas Interface; Wiley: New York, 1966. (5) Nagurama, T.; Kamata, S.; Toyozawa, K.; Ogawa, T. Ber. Busenges. Phys. Chem. 1990, 94, 87. (6) Ohta, N.; Tamai, N.; Kuroda, T.; Yamazaki, T., Nishimura, Y.; Yamazaki, I. Chem. Phys. 1993, 177, 591. (7) Kuhn, H. Pure Appl. Chem. 1965, 11, 345. (8) Kuhn, H. Naturwissenschaften 1967, 54, 429. (9) Roberts, G. G.; Pande, K. P.; Barlow, W. A. Proc. Inst. Electron. Eng., Part I 1978, 125, 169. (10) Gantt, E. Annu. Rev. Plant. Physiol. 1981, 32, 327. (11) Glazer, A. N. Annu. Rev. Biophys. Chem. 1985, 14, 37. (12) Yamazaki, I.; Ohta, N.; Yoshinari, S.; Yamazaki, T. Microchemistry, Spectroscopy and Chemistry in Small Domains; Masuhara, H.; De Schryver, F. C.; Kitamura, N.; Tamai, N., Eds; North-Holland Elsevier Science B. V.: Amsterdam, 1994; p 431. (13) Knox, R. S. In Primary Processe in Biosynthesis; Barber, J. Ed.; Elsevier: Amsterdam, 1977; Vol. 2, p 55.
and photoelectric properties.5,14-16 However, in LB films as well as in other artificial molecular assemblies (e.g., vesicles, micelles, etc.), the in-plane organization is not yet completely understood on a microscopic and submicroscopic level. Since the 1960s17 and as demonstrated by the pioneering work by Kuhn,1,2 direct excitation energy transfer (DET) is a very efficient method to deal with the problems concerning the average structure of monolayer assemblies on scales e100 Å.16-22 In LB films where the molecular diffusion is sufficiently slow compared with the DET process, the Fo¨rster mechanism is often prevalent for excited singlet states.15,23 The expression for the fluorescence decays derived by Fo¨rster were obtained under an assumption of the random and uniform distribution of molecules. However, in several low-dimensional systems, deviations from those expressions have been observed24-30 (14) Roberts, G. Sensors Actuators 1983, 4, 131. (15) Nishiyama, Y.; Azuma, T.; Obata, N., Kasatani, K.; Sato, H. J. Photochem. Photobiol. A: Chem. 1991, 59, 341. (16) Drake, J. M.; Klafter, J.; Levitz, P. Science 1991, 251, 1574. (17) Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci., U.S.A. 1967, 58, 719. (18) Levitz, P.; Drake, M. Phys. Rev. Lett. 1987, 58, 686. (19) Pines, D.; Huppert, D.; Avnir, D. J. Chem. Phys. 1988, 89, 1177. (20) Pines, D.; Huppert, D. J. Phys. Chem. 1987, 91, 6569. (21) Dewey, T. G.; Datta, M. M. Biophys. J. 1989, 56, 415. (22) Tamai, N.; Yamazaki, T.; Yamazaki, I.; Mizuma, A.; Mataga, N. J. Phys. Chem. 1987, 91, 3503. (23) Bohn, P. W. Annu. Rev. Phys. Chem. 1993, 44, 37. (24) Ballet, P.; Van der Auweraer, M.; De Schryver, F. C.; Lemmetyinen, H.; Vuorimaa, E. J. Phys. Chem. 1996, 100, 13701. (25) Tamai. N.; Yamazaki, T.; Yamazaki, I. Chem. Phys. Lett. 1988, 147, 25. (26) Yamazaki, I.; Tamai, N.; Yamazaki, T. J. Phys. Chem. 1990, 94, 516. (27) Kemnitz, K.; Murao, T.; Yamazaki, I.; Nakashima, N.; Yoshihara, K. Chem. Phys. Lett. 1983, 101, 337. (28) Kemnitz, K.; Tamai, N.; Yamazaki, I.; Nakashima, N.; Yoshihara, K. J. Phys. Chem. 1987, 91, 1423. (29) Liu, Y. S.; Li, L.; Ni, S.; Winnik, M. A. Chem. Phys. 1993, 177, 579.
S0743-7463(97)00787-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/06/1998
Cyanine Dye Distribution in Monolayer Determined by Fluorescence
at low dye concentration and were attributed to an inhomogeneous distribution of the donor and/or acceptor dyes in the monolayer24-26 or to a distribution of decay rates or conformations of flexible dyes.25,27-30 At high dye concentration, other deviations from the Fo¨rster type decay can occur due to exciton hopping.31-36 To elucidate the inhomogeneous structure of LB films, spectroscopic tools with spatial resolutions are indispensable. Optical microscopy, in particular scanning confocal fluorescence microscopy,37-40 is now widely used in biological disciplines. Despite the enormous interest this technology holds, to our knowledge, no studies on the LB film structure by confocal fluorescence microscopy have been reported. The aim of the present work is to elucidate the relation between the two-dimensional molecular distribution in LB film revealed by fluorescence confocal microscopy and the two-dimensional intralayer excitation energy transfer. In the present work, a cyanine dye, the N,N′-dioctadecyl thiacyanine molecule (MBT), was incorporated in LB films of fatty acid or phospholipid, where an inhomogeneous dispersion of other dye molecules had been previously deduced from DET studies.15,21,24-26,41 We confirmed that the deviations of the time-resolved data from the Fo¨rster type law do not result from a distribution of decay rates but are associated with an inhomogeneous structure of the LB films due to a phase separation. The correlation between the spatially unresolved data and the scanning confocal fluorescence measurements provide information of the composition of each phase. Experimental Section Materials and Methods. MBT was prepared according to Sondermann42 and was recrystallized twice from acetic acid. The surfactants arachidic acid (Aldrich P. A.) and dipalmitoyl phosphatidic acid (DPPA; Sigma Chemical Company) were used as received without further purification. Stock solutions of MBT (10-4 M) in CHCl3 and arachidic acid (10-3 M) in CHCl3 were prepared separately. To obtain a good solubilization of DPPA, we prepared a 5 × 10-4 M solution in a 97:3 CHCl3/CH3COOH mixture. All solvents used were of spectroscopic grade. Sample Preparation. Mixtures of MBT and arachidic acid or DPPA were spread onto the surface of the water subphase (Milli-Q) containing 5 × 10-4 M Cd(ClO4)2, 6H2O. The concentration of dye in those mixtures varied from 0.5 to 6 mol %. The subphase conditions were adjusted to a temperature of 19.5 °C and the subphase pH was 5.8. As hydrophilic substrata, quartz slides cleaned as previously described were used.43 (30) Hayashi, T.; Okuyama, T.; Ito, S.; Yamamoto, M. Macromolecules 1994, 27, 2270. (31) Blumen, A.; Manz, J. J. Chem. Phys. 1979, 71, 4694. (32) Loring, R. F.; Andersen, H. C.; Fayer, M. D. J. Chem. Phys. 1982, 76, 2015. (33) Huber, D. L. Phys. Rev. B 1979, 20, 2307. (34) Feodorenko, S. G.; Burshtein, A. I. Chem. Phys. 1985, 98, 341. (35) Willig, F.; Blumen, A.; Zumofen, G. Chem. Phys. Lett. 1984, 108, 222. (36) Zumofen, G.; Blumen, A. J. Chem. Phys. 1982, 76, 3713. (37) Hall, A.; Browne, M.; Howard, V. Proceedings RMS 1991, 26, 63. (38) Webb, R. H. Rep. Prog. Phys. 1996, 59, 427. (39) Ghiggino, K. P.; Spizzirri, P. G.; Smith, T. A. Microchemistry, Spectroscopy and Chemistry in Small Domains; Masuhara, H.; De Schryver, F. C.; Kitamura, N.; Tamai, N., Eds; North-Holland Elsevier Science B. V.: Amsterdam, 1994; p 197. (40) Sasaki, K.; Koshioka, M. Microchemistry, Spectroscopy and Chemistry in Small Domains; Masuhara, H.; De Schryver, F. C.; Kitamura, N.; Tamai, N., Eds; North-Holland Elsevier Science B. V.: Amsterdam, 1994; p 185. (41) Ballet, P. Ph.D. Thesis, K. U. Leuven, Leuven, 1995. (42) Sondermann, J. Liebigs Annal. Chem. 1971, 749, 183. (43) Verschuere, B.; Van der Auweraer, M.; De Schryver, F. C. Thin Solid Films 1994, 244, 995.
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The multilayer systems were prepared on a KSV 5000 ALT trough by the LB method. The monolayers were compressed at a speed of 5 mN/m/min until a surface pressure of 30 mN/m was reached. The monolayers were allowed to equilibrate for 10 min before deposition on the substrata and were deposited at a constant surface pressure of 30 mN/m. The dipping speed was 5 mm/min for pure monolayers of Cd-arachidate (AA) or DPPA, 4 mm/min for mixed monolayers of DPPA and MBT, and 2 mm/ min for mixed monolayers of AA and MBT. During the deposition, the compression speed was fixed to 3 mN/m/min (forward) and 1.5 mN/m/min (backward). The deposition ratio amounts to 0.94 ( 0.05 regardless the composition of monolayer. Two different monolayer assemblies were studied. The first one (monolayer system) consisted of a mixed monolayer of MBT and AA or DPPA deposited on the top of four layers of sheer AA or DPPA. In the second type (multilayer system), the hydrophilic quartz plates were covered with four layers of sheer AA or DPPA before deposition of two mixed monolayers of MBT and AA or DPPA in a tail-to-tail arrangement followed by two layers of sheer AA or DPPA. The deposition of the two mixed monolayers followed by two layers of sheer AA or DPPA was repeated five times for the 6 mol %, 4 mol %, and 2 mol % concentrations and seven times for the 1 mol % and 0.5 mol % concentrations. This tail-to-tail arrangement of the mixed monolayers avoids the interlayer dimer formation between chromophores in neighboring monolayers and is expected to strongly reduce the interlayer energy transfer. Apparatus. The absorption spectra were recorded with a Lambda 6 spectrophotometer. A uncoated quartz slide was used as reference. The steady-state fluorescence emission and excitation spectra were recorded on a Spex Fluorolog spectrofluorimeter at an angle of 26° with the incoming light that impinged along the normal of the sample. The spectra were obtained under reduced pressure (1 Torr) to prevent photooxidation. Time-resolved fluorescence studies with excitation at 400 nm were performed by the time-correlated single-photon counting method.44 To obtain time-resolved fluorescence decays of MBT deposited on solid substrata, a sample compartment that could be evacuated to 1 Torr was developed. The angle of incidence of the vertically polarized excitation laser pulses on the quartz substrata was ∼25° to the normal and detection was performed at the other side of the substrata at a right angle to the direction of the excitation beam. The fluorescence was observed through a Glan Thompson polarizer set at the magic angle. In this way, fluorescence polarization or depolarization could not influence the decays. A cutoff filter (Schott-420 nm) screened scattered laser light. The fluorescence was detected by a combination of a polychromator and a multi-anode microchannel plate (Hamamatsu R 3839U-07). This microchannel plate contains a set of 8 linear anodes with a width of 1.5 mm and the dispersion of the polychromator (constructed by IBH with a grating of type 446.31 of American Holographic) amounted to 20 nm per mm, so the fluorescence obtained in a band of 30 nm is collected on each of the anodes. Hence, each anode integrates the fluorescence decays over a bandwidth of 30 nm. Only the decays obtained for the wavelength window 440-470 nm were analyzed. At longer wavelengths, the fluorescence will be due to some extent to dimer emission and the decays must be described by expression that are more complex than eqs 5-8 (cf. infra). The signal from the anodes is amplified by a “Philips Octal Preamplifier” and sent to a discriminator (Tennelec TC 454). After being processed by a “5000MXR IBH Mixer Router”, the signals were used as start pulse in a time to amplitude converter (TAC), (ORTEC567) which receives its stop pulse from a photodiode (EGG ORTEC934) on which a part of the excitation pulse impinges. The signals from the TAC are digitized using a “8192 Wilkinson 100Mhz ADC” and stored in different segments of a multichannel analyzer, depending on their parent cathode. In this way, the fluorescence decays of different wavelength regions can be obtained simultaneously. Laser pulses scattered from a MBT/MeOH solution (lifetime of 18ps) in a 1-cm path length cell were used to generate the instrument response function. The resulting difference in optical path with the quartz (44) O’Connor, D. V.; Phillips, D. In Time-Correlated Single Photon Counting; Academic: New York, 1984.
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Figure 1. Absorption spectra of MBT in DPPA and AA matrixes at different mixing ratios. The spectra are recorded on multilayer systems. substrata is taken into account in the decay analysis by introducing a “shift” parameter that does not exceed 1.24 channels (which corresponds to 36 ps). The fluorescence decays were analyzed, either individually or globally, by iterative reconvolution using a Marquardt algorithm. The fit parameters obtained from either the single curve analysis or the global analysis are similar, so only the results of the global analysis of the fluorescence decays will be presented. It is now well-established that the global analysis leads to more accurate fit parameters than the single curve analysis.45 In the analysis of the fluorescence decays, the presence of scattered light was taking into account. The ratio amplitude of scattered light to I(0), the fluorescence intensity at time 0, is indicated in the tables. The stationary confocal fluorescence measurements have been performed on a scanning confocal fluorescence microscope, as described elsewhere.46 The laser excitation light at 420 nm was reflected by a dichroic mirror (Chroma-435 nm or Chroma-460 nm) and focused onto the sample by an oil-immersion objective with a magnification and a numerical aperture of 60 and 1.4, respectively. The resultant fluorescence is collected and isolated from scattered excitation light using the dichroic mirror and an appropriate blocking filter (Chroma-435 nm or Chroma-475 nm). Each confocal images was accumulated 20 times.
formation of dimers.2 As shown in Figure 1 even at low mixing ratios dimers are observed. The position of the maximum at 432 nm does not depend significantly on the mixing ratio, whereas the maximum at 413 nm is blueshifted from 413 nm at 0.5 mol % to 408 nm at 6 mol % in both DPPA and AA matrixes upon increasing the concentration of MBT. This shift suggests the onset of the formation of larger aggregates. However, because of the broad features of the spectra, it is difficult to make a distinction between the absorption spectrum of dimers and that of aggregate. Nevertheless, as the shift remains very modest, we will assume that most of the aggregates will be dimers for the concentration range we investigated. From these absorption spectra, the monomer and dimer fractions of MBT molecules incorporated in LB film can be determined, assuming the conservation of the overall oscillator strength.47,48 The absorbance originating from the monomers and dimers of MBT molecules according to the LB law is given by
Results
The subscripts M and D refer to the monomers and dimers, respectively. The total concentration of MBT molecules (Ctot) is related to the concentration of monomers (CM) and dimers (CD) (in mol dm-3) as Ctot ) CM + 2CD while l corresponds to the thickness of the mono- or multilayer system. abs The monomer (f abs M ) and dimer (f D ) fractions, as determined from the absorption spectra, will be defined as follows:
Surface Pressure-Area Isotherms. Because of the small amounts of MBT in the mixed monolayers, the surface pressure isotherms of the mixed monolayers of MBT and AA and of MBT and DPPA are within the experimental error similar to the isotherms of the monolayers of respectively, sheer AA2 and sheer DPPA.45 Steady-State Measurements. Because of the incomplete match of the sample and the reference, the absorption spectrum obtained for a single monolayer cannot be distinguished from the noise. Consequently, to obtain the best signal-to-noise ratio, the absorption spectrum from MBT in mixed monolayers has been determined on the basis of spectra recorded for the multilayer systems. On the other hand, the fluorescence emission and the fluorescence excitation spectra collected from samples containing one mixed monolayer are characterized by a good signal-to-noise ratio. These spectra have identical features as those obtained for the multilayer systems. In addition to a maximum at 432 nm corresponding to the MBT monomer, a second maximum appears at 413 nm in the absorption spectra that is attributed to the (45) Van der Auweraer, M.; Ballet, P.; De Schryver, F. C.; Kowalczyk, A. Chem. Phys. 1994, 187, 399.
Atot(ν) ) M(ν)CMl + D(ν)CDl
f abs M ) CM/Ctot
f abs D ) CD/Ctot
(1)
abs f abs M + 2f D ) 1 (2)
The first-order approximation of the exciton theory implies
∫D(ν) dν ) 2∫M(ν) dν
(3)
So, the monomer and dimer fractions can be calculated (46) Hofkens, J.; Latterini, L.; Vanoppen, P.; Faes, H.; Jeuris, K.; De Feyter, S.; Kerimo, J.; Barbara, P. F.; De Schryver, F. C.; Rowan A. E.; Nolte, R. J. M. J. Phys. Chem., B 1997, 101, 10588. (47) MacRae, E. G.; Kasha, M. The Molecular Exciton Model In Physical Processes in Radiation Biology; Academic: New York, 1964. (48) Kasha, M.; Rawls, H. R.; El Bayoumi M. A. Pure and Appl. Chem. 1965, 11, 371.
Cyanine Dye Distribution in Monolayer Determined by Fluorescence
Langmuir, Vol. 14, No. 18, 1998 5175
Figure 2. Fluorescence spectra of a mixed monolayer of MBT and DPPA and of a mixed monolayer of MBT and AA at different mixing ratios and upon excitation at 432 nm. The spectra are normalized to a constant area equal that was put equal to one. Table 1. Fraction of MBT Molecules Present as Dimers (f abs D ) and Relative Fluorescence Quantum Yields (O/ O0.5mol %) of MBT in AA and DPPA Matrices as a Function of the Mixing Ratio at Room Temperature DPPA
AA
mol % MBT
a f abs D
φ/φ0.5mol%
a f abs D
φ/φ0.5mol%
6 4 2 1 0.5
0.21 ( ( 0.02 0.23 ( 0.02 0.19 ( 0.02 0.22 ( 0.05
0.41 0.40 0.49 0.54 1
0.22 ( ( 0.02 0.23 ( 0.02 0.20 ( 0.04 0.10 ( 0.07 0.10 ( 0.07
0.32 0.42 0.27 0.78 1
a
The values of f abs D are determined from the absorption spectra.
from the following relations combining eqs 1, 2, and 3
f abs M )
∫AM(ν) dν ∫Atot(ν) dν
1 1 abs f abs D ) (1 - f M ) ) 2 2
∫AD(ν) dν ∫Atot(ν) dν
(4)
where AM(ν) and AD(ν) represent, respectively, the absorption spectrum of the monomers and the dimers of MBT molecules. As the molar extinction coefficient of the monomer of MBT in a LB film, we used that of MBT in methanol at high dilution multiplied by 3/2. Indeed, to take into account the restricted orientation of the dye in the LB film compared with the isotropic orientation of the dye in solution, a geometrical factor has to be considered. In an LB film, half of the dye molecules have their transitions dipoles oriented parallel to the electric field of the polarized excitation light, whereas, in an isotropic solution, this is only the case for one-third of the dye molecules. Consequently, the molar extinction coefficient of the monomer of MBT in LB film corresponds to 3/2 times of that of MBT in solution. Under those conditions, using eq 4 and assuming that at the maximum at 432 nm there is no dimer absorption, the monomer and dimer fractions of MBT molecules in LB film have been estimated (Table 1) and we observe that at low dye concentration, a significant fraction of the MBT molecules are already present as dimers in the LB film. Despite the experimental error on the values, most of which is due to an incomplete match of the sample and the reference, leading to an inaccurate determination of the baseline,
the dimer fraction is constant over the concentration range studied and amounts to 0.20 ( 0.03. This means that even if the dimers have a weak long-wavelength absorption maximum, this will not invalidate our assumption that we could neglect at 432 nm the dimer absorption versus the monomer absorption. As the mixing ratio of the dye and the matrix increases, a red-shift of the emission spectra (Figure 2) and a decrease of the quantum yield (Table 1) have been observed for MBT in mixed monolayers. This decrease amounts to 50% for both matrixes. The latter phenomena suggests the occurrence of energy transfer from monomers to dimers. The red-shift is more pronounced in the AA matrix in which the emission maximum shifts from 469 nm at 0.5 mol % to 486 nm at 4 and 6 mol %, whereas in the DPPA matrix it shifts from 471 to 481 nm over the same concentration range. Time-Resolved Fluorescence Measurements. The time-resolved fluorescence decays obtained upon excitation at 400 nm have been recorded for MBT in the monolayer and the multilayer systems over the wavelength window 440-470 nm. These decays are clearly not monoexponential even at lowest concentration used (Figure 3). As expected the decays depend on the mixing ratio, the decays being more rapid at higher MBT concentration. Assuming that the decrease of the fluorescence quantum yield is due to Fo¨rster energy transfer from monomers to dimers in homogeneous or self-similar systems, one could expect to analyze the fluorescence decays according to the following well-known decay law derived for Fo¨rster type excitation transfer49-51
I(t) ) I(0) exp[-(t/τD) - γdh (t)dh /6]
(5)
where τD represents the fluorescence decay time of the donor in the absence of acceptors, the parameter is a function of the critical energy transfer distance R0 and the number of the acceptors per unit area, and represents the (fractal) dimension of the distribution of the acceptors. When, as assumed for the mono- and multilayers of MBT and AA or DPPA, the direct energy transfer from monomers to dimers can occur only in the plane of the (49) Klafter, J.; Blumen, A. J. Phys. Chem. 1984, 80, 875. (50) Klafter, J.; Blumen, A. J. Lumin. 1985, 34, 77. (51) Klafter, J.; Blumen, A. Chem. Phys. Lett. 1985, 119, 377.
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Figure 3. Influence of the mixing ratio on the fluorescence decays of a mixed monolayer of MBT and DPPA and of a mixed monolayer of MBT and AA upon excitation at 432 nm. The decays are recorded by integrating the emission obtained between 440 and 470 nm. The decay profile of the scattered light is also shown on the figure. Table 2. Global Analysis of the Fluorescence Decays of MBT in AA and DPPA Momolayers According to Equation 8 mol % MBT
γ2 (ns-1/3)
6 4 2 1 0.5
1.490 ( 0.020 1.189 ( 0.007 1.950 ( 0.009 1.083 ( 0.009 0.739 ( 0.008
6 4 2 1 0.5
2.04 ( 0.03 2.13 ( 0.03 1.51 ( 0.03 1.25 ( 0.04 0.97 ( 0.04
χ2
scatter
calc φcalc/φ0.5mol%
MBT/AA 0.053 ( 0.003 0 0 0 0
1.05 1.12 1.23 1.22 1.13
0.49 ( 0.01 0.47 ( 0.01 0.45 ( 0.01 0.45 ( 0.02 0.57 ( 0.01
0.57 0.62 0.31 0.69 1
MBT/DPPA 0.0570 ( 0.003 0.0413 ( 0.003 0.0872 ( 0.007 0.085 ( 0.01 0.074 ( 0.02
1.08 1.26 1.05 1.12 1.10
0.39 ( 0.01 0.46 ( 0.01 0.51 ( 0.01 0.49 ( 0.02 0.53 ( 0.02
0.45 0.39 0.70 0.82 1
1-R
a The decay time of the monomer in the absence of quenching dimers, τ , is linked over the concentration range studied. The decays D are recorded on monolayer system and the emission was integrated between 440 and 470 nm. The fluorescence quantum yields φcalc are 2 calculated according to eq 9. For MBT/AA, τD and χg amount to 2.92 ( 0.06 ns and 1.14, respectively. For MBT/DPPAA, τD and χ2g amount to 2.84 ( 0.15 ns and 1.12, respectively.
monolayers, eq 5 becomes, for homogeneous systems (d h ) 2):
I(t) ) I(0) exp[- (t/τD) - γ2(t)1/3]
(6)
γ2 ) Γ(2/3)πσAR02(τD)-1/3
(7)
where
The parameter designates the Euler-gamma function. For systems showing inhomogeneity on a scale much larger than R0, an additional monoexponential decaying term attributed to donor molecules not affected by the energy transfer had sometimes to be included in the previous equations.52,53 In this case, the fluorescence decay has to be fitted to following equation:
over different concentrations. The decay parameters obtained for the monolayers are reported in Table 2. Similar fit parameters were obtained for the multilayers. It was checked that the introduction of a “scatter” parameter, which corrects as well for the scattered light as for digitalization errors,54-57 does not mask a fast decay component of the excited states. Indeed, the decrease of the fluorescence quantum yield upon increasing the mixing ratio, as determined from eq 9 where the parameters R, τD, and γ2 are deduced from the analysis of the time-resolved decays, parallels that obtained from the analysis of the stationary fluorescence spectra (Table 1).
φcalc ) kf
∫0+∞{R exp[- (t/τD) - γ2(t)1/3] +
(1 - R) exp[-t/τD]} dt (9)
1/3
I(t) ) I(0){R exp[-(t/τD) - γ2(t) ] + (1 - R) exp[-t/τD]} (8) The global analysis of the fluorescence decays of MBT in the monolayers and multilayers in the framework of eq 8 has been performed linking the decay time of the monomer in the absence of quenching dimers, τD, (52) Hauser, M.; Klein, U. K. A.; Go¨sele, U. Z. Z. Phys. Chem. N. F. 1976, 101, 255. (53) Baumann, J.; Fayer, M. D. J. Chem. Phys. 1986, 85, 4087.
The linked parameter τD amounts to 2.90 ns for the wavelength range between 440 and 470 nm for both (54) Periasamy, N. Biophys. J. 1988, 54, 961. (55) Holtom, G. R. Artifacts and diagnostics in fast fluorescence measurements, In Time-Resolved Laser Spectroscopy in Biochemistry II. SPIE 1990, 1204, 2. (56) Kowalczyk, A.; Boens, N.; Van der Auweraer, M.; De Schryver, F. C., submitted for publication. (57) The digitalization errors could be due to the fact that the actual IRF are a continuous function of time whereas the data we obtained are integrals over channel.
Cyanine Dye Distribution in Monolayer Determined by Fluorescence
Figure 4. Influence of the total dye concentration (σtot) of MBT in AA and DPPA matrixes on the parameter γ2, obtained by global analysis in the framework of eq 8. Table 3. Comparison between the Values of the Parameter γ2 Obtained at a Concentration of 0.5 mol % or Various Dyes Incorporated in a DPPA Matrix with the Ratio of the Singlet Decay Time of the Dyes in Solution and in the LB Film dye rhodamine B in solution τD ) 3 ns24 in LB film τD ) 3 ns24 MBT in ethanol, τD ) 18 ps72 in LB film, τD ) 3 ns crystal violet in solution, τD ) 2 ps60-63 in LB film, τD ) 2.7 ns41,61,63
τD(LB film)/ τD(solution) 1
γ2 (ns-1/3) at 0.5 mol % 0.324
166
0.9
1325
241
matrixes (AA and DPPA). The value of τD is of the same order of magnitude as the fluorescence lifetime obtained for the analogue dye, N,N′-dioctadecyl oxacyanine, which amounts at a concentration of
