J. Phys. Chem. 1995,99, 14931-14941
14931
Solvent Relaxation around the Excited State of Indole: Analysis of Fluorescence Lifetime Distributions and Time-Dependence Spectral Shifts Michel Vincent and Jacques Gallay* L. U.R.E. Laboratoire pour 1'Utilisation du Rayonnement Electromagnttique, Universitt Paris-Sud, Batiment 2090, 91405-0rsay, France
Alexander P. Demchenko Department of Biophysics, A. V. Palladin Institute of Biochemistry, Kiev, 252030, Ukraine Received: March 9, 1995; In Final Form: August 4, 1995@
Analysis of the fluorescence lifetime distributions of indole in polar protic solvents by the maximum entropy method (MEM) has allowed to obtain relatively narrow peaks, which originate first from the main emission decay component and second from additional short components which are due to spectral kinetics processes. These latter components are characterized by positive amplitudes at short emission wavelengths and by negative amplitudes (with the same mean time component value) at long-wavelength edge of the fluorescence spectrum. For both positive and negative components, the effect of red-edge excitation is strongly pronounced: they sharply decline or even disappear if the excitation is performed at the long-wavelength edge of the absorption spectrum (300 nm). As expected, these components are very sensitive to temperature. The observed relaxational component shifts to longer values as the temperature decreases. The variation is linear in Arrhenius coordinates and results in activation energies of 7.5 kcdmol. The shifts of emission spectra as a function of time (TRES) have allowed to obtain relaxation times in the same range as observed in lifetime distributions. The C(t) function defined by using the barycenters of the emission spectra can be described by MEM as a sum of two or three discrete species depending on the temperature. The longest one displays a value similar to that of the longest longitudinal relaxation time of isobutyl alcohol as determined by dielectric measurements in the same temperature range. The excited state stabilization energy is around 3.7 kcal/mol. This set of results is consistent with a mechanism of general dielectric solvent relaxation rather than formation of binary excited state complexes in the time range studied.
Introduction Dielectric relaxation of the polar environment of excitable molecules is an important process, which results in substantial shifts of their fluorescence emission spectra to lower energies.'-' These relaxations may also provide a dynamic control of the rates of several kinds of excited state reaction^.^.^ The first manifestations of these relaxation processes were obtained in steady-state experiments by shifts of the fluorescence emission spectra as a function of temperature from low-temperature unrelaxed form to high-temperature relaxed and in timeresolved fluorescence experiments by emission wavelength dependence of the average excited state lifetime ( T ) . ~ , ~ Time-resolved emission spectra (TRES) showed a gradual shift of the spectral envelope to longer wavelengths as a function of time.293.7 From the time evolution of the spectral shift, the solvent relaxation rate(s) could be calculated. The simple theory based on the Debye model of relaxation suggested a singleexponential time dependence of the spectral shifts.' Usually, the values of dipolar relaxation times obtained by the fluorescence method have been shown to correspond to the values of the dielectric relaxation times, to, as measured by dielectric dispersion method (with the account of correction factor') and to longitudinal relaxation times, TI,,defined as TL = (E-/EO)TD*D~'~. Recent experiment^^.^.'^ have demonstrated, however, that the situation is more complex. The evolution with time of the barycenter of the emission spectrum was rarely observed to follow single-exponential decays.' This might be due both to
* To whom correspondence should be addressed. @
Abstract published in Advance ACS Abstracts, September 15, 1995.
0022-365419512099-14931$09.0010
a change of the spectrum shape in the course of relaxation (and, in particular, increase or decrease of its width) and to more complex mechanisms of relaxation which may imply the molecular structure of the solvent and probe-solvent specific interactions as well, since they will induce deviation from the continuum model. For instance, the bulk solvent molecules and the closest neighbors to the chromophore may display different relaxation properties2 depending on the relative strengths of the solvent-solvent and solute-solvent interactions. Moreover, the solvent molecules are usually nonsymmetric dipoles and display different relaxation rates along their different axes.9 Most importantly, intermolecular rearrangements of the chromophore,' formation of specific complexes in the excited state (exciplexes),I2and reorganization of chromophore-solvent hydrogen bondslOa-b may result in temporal shifts of fluorescence spectra, which can be very similar to that due to dipolar relaxations. Therefore, the development of new approaches for the analysis of time-resolved spectral data is still necessary to obtain specific information from these data. Up to now, a number of authors have considered theoretical analysis of the solvent relaxation processes accounting for the existence of distributions of chromophore-solvent interaction energies, which always exists in solutions and results in inhomogeneous broadening of the spectra.I3-l5 Dependent on the relaxation rate, this broadening may be both static and dynamic. It was demonstrated, however, that by shifting the excitation wavelength to the red edge of the absorption spectrum, particular minor species within this distribution may be phot~selected.'~-'~ Their interaction energy with the solvent molecules is stronger than that of the mean of the population 0 1995 American Chemical Society
Vincent et al.
14932 J. Phys. Chem., Vol. 99, No. 41, 1995 and close to that of the relaxed state. As expected, this socalled red-edge effect allows not only to select the specific excited state populations differing in their interactions with the surrounding solvent molecules but also to study the dynamics of these interactions. The position of the steady-state fluorescence emission spectra,I6-l7 the extent and even the direction of motion of the time-resolved spectra,I7the excited state energy transfer,20segmental mobility,21and electron transfer22can be changed as a result of this type of photoselection. This allows to obtain information on the dynamic behavior and excited state reactivity of the ensemble of chromophore-solvent interactions. It was suggested that the observation and analysis of the rededge effects may allow to reach better understanding of the relaxation mechanism, to discriminate dipolar relaxations from other reactions occurring in the excited state, and to determine the relaxation rate value^.'^^'^ The site-selective spectroscopy at the red edge may be easily combined with time-resolved measurement^,'^ and though the first attempts were promising (in particular, in determination of the dielectric relaxation rates in proteins23),this combined approach is only at the beginning of its development and exploration. In this work we have focused on the study of fluorescence spectroscopy of indole in polar solutions, a molecule which is extensively studied,24due to the importance of its derivative tryptophan for fluorescence studies of biological systems and proteins in particular.Is We report on the first application in the studies of solvent dielectric relaxations of excited state lifetime distributions analysis by the maximum entropy method (MEM),25-28and on the dependence of the observed effects on excitation wavelength (the red-edge effect) for indole. For these purposes, the fluorescence lifetime distributions were obtained for indole in different polar solvents (isobutyl alcohol, ethanol, and water) as a function of temperature at different emission wavelengths at the mean and red-edge excitations. Timeresolved emission spectra (TRES) were also constructed and analyzed in order to measure the amplitude and the kinetics of the time-dependent fluorescence shift (TDFS). This phenomenon is important to explore since in fact it reveals the existence of the relaxation process of the solvent cage around the excited state of the probe.
excited state lifetime the instrument is able to resolve has values of 20-30 PS. Data Analysis of Fluorescence Lifetime Distributions. Analysis of the fluorescence intensity decay data as a sum of 150 exponentials was performed by the maximum entropy m e t h ~ d . ~The ~ - ~programs ~ uses MEMSYS 5 (MEDC Ltd., U.K.) as a library of subroutines. Optionally, MEMSYS 5 can handle a 150-dimensioned vector, without any a-priori assumption on each amplitude sign. This option was used in cases where the classical analysis with only positive amplitudes did not provide good results in terms of x2 values and shape of the deviation function of the weighted residuals. Since we are working with polarized light, the total intensity decay is reconstructed by adding the parallel and twice the perpendicular components:
Experimental Section
where Ikcalcand Tkobs are the kth calculated and observed intensities. ak2,the variance of the kth point, is equal to q v v 2 4/320k,vh2.3’ M is the total number of observations. The center (z) of a single class j of lifetimes over the a(zi) distribution was defined as
Chemicals. Indole and N-methylindole were purchased from Sigma. Ethanol and isobutyl alcohol were from Merck (spectroscopic grade). Fluorescence Lifetime Measurements. Fluorescence intensity decays were obtained by the time-correlated single photon counting technique29from the polarized components Ivy(t) and on the experimental setup installed on the SBI window of the synchrotron radiation machine Super-ACO (Anneau de Collision d’Orsay), which has been described elsewhere.30 The storage ring provides a light pulse with a fullwidth at half maximum (fwhm) of -500 ps at a frequency of 8.33 MHz for a double bunch mode. A Hamamatsu microchannel plate R1564U-06 was utilized to detect the fluorescence photons. Data for Zvv(t) and Ivh(t) were stored in separated 2K memories of a plug-in multichannel analyzer card (Canberra) in a DESKPRO 286E microcomputer (Compaq)’. The automatic sampling of the data was driven by the microcomputer. The instrumental response function was automatically collected each 5 min by measuring the scattering of a glycogen solution during 30 s at the emission wavelength, in alternation with the parallel and perpendicular components of the polarized fluorescence decay, which were cumulated during 90 s. The time resolution was usually in the range of 10-20 ps per channel. The shortest
where EA(f)is the temporal shape of the excitation light pulse, * denotes a convolution product, Bo, is a correction factor3’ taking into account the difference of transmission of the polarized light components by the optics, and a(z)is the lifetime distribution given by
The recovered distribution a(z)which maximizes the entropy function S is ~ h o s e n : ~ ~ . ~ ~
In this expression, m ( t ) is the starting model. In every analysis, a flat map over the explored (t) domain was chosen for m ( t ) ,since no a priori knowledge about the final distribution was available. 28 The analysis was bound by the x2 constraint:
(4) k= I
+
the summation being performed on the significant values of the a(tJfor the j class. C, is the normalized contribution of the lifetime class j. The broadeness w, of the distribution was calculated according to
Time-Resolved Spectra: Collection and Analysis. TRES were reconstructed in each experimental conditions from -20 individual decays as a function of the emission wavelength from 305 or 310 nm up to 400 nm (bandwidth 5 nm) with a 5 nm step. The decays were cumulated up to 1 ,04 counts in the peak channel. Each individual curve was fit with the MEM program using the negative amplitude option. The integral of each decay curve was normalized to the corresponding steady-state fluorescence emission wavelength recorded on the same instrument
Solvent Relaxation around the Excited State of Indole
J. Phys. Chem., Vol. 99, No. 41, 1995 14933
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Figure 2. MEM recovered lifetime distribution of indole in water at 20 "C. Excitation wavelength: 280 nm (bandwidth: 5 nm); emission wavelength: 350 nm (bandwidth: 10 nm). t = 4.57 ns; x2 = 1.14.
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Figure 1. Recovery of decay time constant distributionsby MEM from simulated data. (Upper curve) Starting values: 51= 80 ps, al= 0.250, t2 = 3.50 ns, a2 = 0.750; recovered values: tl = 76 ps (w = 20 ps), al= 0.256, t2 = 3.50 ns (w = 0.038 ns), a2 = 0744. x2 = 1.006. (Lower curve) Starting values: tl = 80 ps, al= -0.50, z2 = 3.5 ns, a2 = 0.50; recovered values: tl = 85 ps (w = 37 ps), al = -0.51, t2 = 3.59 ns (w = 0.62 ns), a2= 0.49. x2 = 0.997. with identical experimental conditions. By collecting the vertical (Ivv)and horizontal (Ivh) fluorescence intensity components and by taking into account the B correction factor,31the calculated impulse fluorescence intensity (expression 1) as well as the steady-state intensity (Ivv 2/3Zvh) are defucro corrected for the difference of transmission of the polarized light components by the optics. Steps of -25 ps were used for the spectral shift construction. For the quantitative description of the spectral shift, the barycenters in frequency were computed from the raw spectra and the full widths at half-peak were calculated from smoothed spectra. The shift function C(r) was defined classically
+
cir,= { V I - V,}/{V0 - V,} where ijt, i j o , and P , are the barycenter values in frequency at time t, 0, and m, respectively. Relaxation times were determined from a MEM analysis of C(r), as a sum of exponential^.^^-^^
(7) Results Recovery of Simulated Data by MEM. The ability of MEM to recover short decay times affected by either a positive or a negative amplitude was tested on simulated data with sets of parameters (ai, ti)in the same range as that experimentally found in the studies described in this work. The injected lifetime parameters were 80 ps and 3.5 ns with relative proportions of 0.290.75 for the positive distributions and -0.50/+0.50 for both allowed negative and positive distributions. The recovered parameters were in both cases very satisfactory (Figure 1). The recovered value of the short time constant (center of the recovered distribution) is different by only 4-5 ps (-5%) from the injected value. MEM analysis provides, however, always broad distributions (characterized by the broadness wj)for short
time constants, especially for negative amplitude distributions. This is first the result of the poorer accuracy of the time constant determination in the time range of a few tens of picoseconds in decay data presenting a set of time constants ranging from picoseconds to several nanoseconds, due to Gaussian noise inherent to the sampling method. Nevertheless, the shortest value that can be recovered with our instrumentation is -20 ps which corresponds to the order of magnitude of the time resolution used in the experiments. Such a rather good accuracy at short times can be obtained with the synchrotron pulse (500600 ps) owing to its high stability (no jitter) and to the fact that its normal time evolution is averaged out by alternative collection of the fluorescence data and of the instrument response function. The recovery of a distribution is also an intrinsic effect of the log t scale. Moreover, when both positive and negative distributionscoexist, the positive long time constant is also recovered as a broader distribution than when only positive distributions are present in the simulated decays. This is exactly what is expected from MEM analysis since this method allows to recover an envelope containing the physical solution. This apparent broadening is due to the fact that the number of physical solutions in the case of negative components is twice as large as in the case of positive components only, the final image being the difference between two images.25 Additional peaks of both positive and negative amplitudes can be observed (Figure 1). They can be due to a compensation phenomenon between the positive and the negative lifetime amplitude spaces for which the algebraic resulting convolution is equal to the instrumental response function. Lifetime Distributions of Indole at Different Emission Wavelengths. A unimodal and very narrow excited state lifetime distribution can be expected if the solvent is rapidly mobile and if the emitted light quanta are collected at the maximum of emission spectrum. Figure 2 demonstrates that, indeed, for indole in water, it is the case: the observed single lifetime distribution is very narrow, close to a single lifetime value (center 4.64 ns). There is no evidence for solvent relaxation around the excited state of indole in water within the allowed time resolution. The rate of the reorientation of the water dipoles (which is faster than picosecond3') is in fact too fast to be detected with our present instrumentation. h ethanol, even at room temperatures, we can observe already the signature of solvent molecular relaxation around the excited state of either indole or N-methylindole, which occurs in the time scale of tens of picoseconds, at the limit of our instrumentation. The lifetime distributions are simply characterized by two positive lifetime populations at short-wavelength emissions, one negative lifetime distribution, and one positive distribution with the same respective barycenters within the experimental errors, at longer emission wavelengths (Figure 3).
14934 J. Phys. Chem., Vol. 99, No. 41, I995 . . . .....
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Figure 3. (a, top) MEM recovered lifetime distribution of indole in ethanol at 20 "C. Upper curve: excitation wavelength 270 nm (bandwidth 10 nm), emission wavelength 310 nm (bandwidth 5 nm). TI = 22 ps (w = 20 ps), a 1 = 0.53,t2 = 3.66 ns (w = 0.12 ns), a2 = 0.47, x2 = 1.03. Lower curve: excitation wavelength 280 nm (bandwidth 5 nm), emission wavelength 370 nm (bandwidth 10 nm). tl = 32 ps (w = 23 ps), al= -0.55,t 2 = 3.67 ns (w = 0.73 ns), a2 = 0.45,x2 = 1.01.(b, bottom) MEM recovered lifetime distribution of N-methyl-indole in ethanol at 20 "C. Upper curve: excitation wavelength 290 nm (bandwidth 5 nm), emission wavelength 310 nm (bandwidth 5 nm). TI = 31 ps (w = 28 ps), a1 = 0.45,t2 = 4.30ns (w = 0.23ns), a2 = 0.55, x2 = 1.18. Lower curve: excitation wavelength 290 nm (bandwidth 5 nm), emission wavelength 400 nm (bandwidth 10 nm). TI = 33 ps (w = 24 ps), a1 = -0.54,t2 = 4.57 ns (w = 1.29 ns), a2 = 0.46,x2 = 1.15.
Such an identity of the fast decay times in the extreme regions of the fluorescence emission spectrum was noticed when the relaxational component is much faster than the fluorescence lifetime.g The long-lived emissions are therefore characterized by a sharp lifetime peak which center values are almost emission wavelength independent for both chromophores. No substantial
differences within the instrument detection limits are observed for the short time constant between indole and N-methylindole which differs from the parent indole in redistribution of electronic charge in both ground and excited states and, probably, in reorganization of hydrogen bonding in the excited state. This may explain the longer excited state lifetime of N-methylindole. The rapid decay and buildup time constant display values of the same order of magnitude as the longitudinal relaxation time value of ethanol calculated from published data (~~2= 0 032.6 ~ p ~ ) . ~ ~ ~ * ~ To obtain a better accuracy on the determination of the rate of the relaxation process, isobutyl alcohol, which is a solvent of higher viscosity than ethanol, has been used in the temperature range of -40 to +20 "C. As in ethanol at 20 O C , the lifetime distribution of indole in isobutyl alcohol, at the same temperature, shows the presence of a single short component in addition to the main peak of emission decay. Its contribution is positive at short-wavelength edge and negative at the long-wavelength edge of the emission spectrum. It displays similar values in these two spectral regions as observed for ethanol (center value obtained for two experiments in the blue edge = 133 f 32 ps; center value for the red edge = 96 f 30 ps) (Figure 4). The long-lived emission is characterized by a single decay time with close values in the two extreme spectral region (center value = 3.37 f 0.02 ns in the blue edge and 3.47 f 0.17 ns in the red edge of the fluorescence emission spectrum). The values of the short decay and buildup time constants are longer than in ethanol and in fair agreement with the longitudinal relaxation time zLcalculated from published data on l - b ~ t a n o l ~but~ .shorter ~ ~ ~ than the published values for isobutyl alcohol.40b Temperature Dependence of the Excited State Lifetimes and of the "Relaxational" Times. As expected, the respective values of each component of the lifetime distribution ("emissional" and "relaxational") display very different thermal variations. The first component is almost thermal insensitive. This effect is frequently observed and is usually explained by an increased probability of thermoactivated radiationless intemal conversion to the ground state.'* The variation of the shortliving relaxational component with temperature is by contrast very strong. In the temperature range of our studies, its value in isobutyl alcohol changes by several orders of magnitude (Table l), which is in line with the data on temperaturedependent variation of dielectric relaxation times of these and similar alcohol solvent^.^*-^ Therefore, we assume that this component arises from dipolar relaxations of the solvent molecules in agreement with our former observations at room temperature. Its variation is linear in Arrhenius representation (Figure 5). As the temperature decreases, the relaxation rate becomes of the same order of magnitude as the fluorescence decay rate (at approximately 225 K for isobutyl alcohol), making more difficult the peak separation. These results demonstrate that in our case, one of the basic assumptions of the Bakhshiev-Mazurenko model' of solvent relaxation is approximately justified, i.e., the separation of the function describing the emission contour as a function of time and energy I(v,f)into two independent contributions, the first one describing the emission decay characterized by the emitting lifetime zf and the second one describing the spectral relaxation characterized by a relaxation time ZR (see Discussion). This allows us to introduce a simple correction to the experimental value Z,I, which accounts for the contribution of emission decay since Z,I is the harmonic mean between the emitting lifetime zf and the true relaxation time ZR. The corrected relaxation time values ZR are presented in Table 1. Only at lower temperatures
Solvent Relaxation around the Excited State of Indole 1
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J. Phys. Chem., Vol. 99, No. 41, 1995 14935 ' '
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Figure 4. (a, top) MEM recovered lifetime distribution of indole in isobutyl alcohol at 20 "C. Upper curve: excitation wavelength 280 nm (bandwidth 5 nm), emission wavelength 315 nm (bandwidth 5 nm). tl = 91 ps (w = 31 ps), al= 0.26, t 2 = 3.36 ns (w = 0.18 ns), a2 = 0.74, x2 = 1.07. Middle curve: excitation wavelength 280 nm (bandwidth 5 nm), emission wavelength 380 nm (bandwidth 10 nm). tl = 75 ps ( w = 62 ps), al = -0.49, t 2 = 3.59 ns (w = 0.70 ns), a2 = 0.51, x2 = 1.06. Lower curve: excitation wavelength 300 nm (bandwidth 5 nm), emission wavelength 400 nm (bandwidth 5 nm). tl = 4.00 ns, x2 = 1.03. (b, bottom) MEM recovered lifetime distribution of indole in isobutyl alcohol at -40 "C. Upper curve: excitation wavelength 280 nm (bandwidth 5 nm), emission wavelength 305 nm (bandwidth 5 nm). tl = 1.90 ns (w = 0.30 ns), a1 = 0.47, t 2 = 4.66 ns ( w = 0.70 ns), a 2 = 0.53, x2 = 1.37. Middle curve: excitation wavelength 280 nm (bandwidth 5 nm), emission wavelength 360 nm (bandwidth 5 nm). t~= 200 ps (w = 80 ps), a1 = -0.05, t 2 = 1.32 ns (w = 0.34 ns), a2 = -0.36, t3 = 4.64 ns (w = 1.04 ns), a3 = 0.59, x2 = 0.90. Lower curve: excitation wavelength 300 nm (bandwidth 5 nm), emission wavelength 400 nm (bandwidth 5 nm). tl = 3.66 ns, x2 = 1.14. TABLE 1: Values of Solvent Dielectric Relaxation Times, ZR (ns), Obtained from MEM Analysis of the Excited-State Lifetime Distributions at Two Emission Wavelengths and from MEM Analysis of the C(t) Function Describing the Time-Dependent Shift of TRES for Indole in Isobutyl Alcohol" lifetime distributions (ns)
-40 -20 +1 +20
3.22 0.67 0.22 0.09
1.85 0.52 0.25 0.08
0.14 (21%) 1.06 (44%) 0.15 (31%) 0.42 (47%) 0.08 (61%) 0.05 (46%)
4.01 (35%) 0.95 (23%) 0.29 (39%) 0.17 (54%)
The values in parentheses represent relative contribution of components in the case of TRES. is the correction significant since both time constants reach similar values. The ZR values obtained at the blue and red edge of the fluorescence emission spectrum are, however, different. From the temperature dependence of ZR,the activation energy of solvent dipolar relaxation can be determined. Its value is around 7.5 kcaVmol for the decay profile at shorter emission wavelength and 8 kcaVmol for that at longer emission wavelength for the temperature range +20 to -40 "C. These values are reasonable in view of our knowledge on activation energy
0.003
0.0035 0.004 1K (K')
0.0045
Figure 5. Arrhenius representation of the emission and relaxational time constants: (0)excited state lifetime measured in the blue edge of the emission spectrum; (*)excited state lifetime measured in the red edge of the emission spectrum; (D) relaxational time constant measured in the blue edge; (0)relaxational time constant measured in the red edge; (0)and (0) time constants obtained by MEM analysis of C(r). of solvent rotational motions and especially in isobutyl alcohol (6.7 kcaVmol).40b Red-Edge Effects on the Relaxational Time Constant, One important feature of dipolar relaxations is clearly observable in the lifetime distributions. If excitation is performed not at the
14936 J. Phys. Chem., Vol. 99, No. 41, I995
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TABLE 2: Values of Fluorescence Emission Lifetimes, ZF (ns) (& Standard Deviation for 2-3 Measurements), as a Function of Excitation and Emission Wavelengths, Obtained from the Corresponding Maxima of the Excited State Lifetime Distributions for Indole in Isobutyl Alcohol lifetime distributions
-40
-20 +1 +20
280 300 280 300 280 300 280 3 0
4.61 f 0.12 4.18 f 0.11 4.03 3.94 3.92 3.94f 0.01 3.37 f0.02 4.03
4.70 f 0.07 3.72f 0.29 4.09 4.06 4.10f0.20 3.40 f0.06 3.47 f 0.17 4.06
maximum of the absorption spectrum (280 nm) but at far red edge in the absorption spectrum (300 nm), the relaxational time constant substantially decreases in amplitude or disappears completely (Figure 4). These observations are in line with the existing knowledge about inhomogeneous kinetics of dielectric relaxation^.^^.'^ In the red edge of excitation, a photoselection of those indole species which interact most strongly with their environment is occurring. For those species, solvent relaxation is not associated with a substantial increase in interaction energy with solvent molecules, and thus, there occurs no significant time-dependent change in the energy of emission quanta.I7-l9 Their steady-state spectra are shifted substantially in the direction of long wavelengths and their time-resolved spectra should not display significant time dependence. Wavelength Dependence of the Excited State Lifetime. The observation of a sharp peak corresponding to the emission time constant in the lifetime distributions allows to determine its value as a function of excitation and emission wavelengths. The existence of a dependence on Lex could be an indication of inhomogeneous decay kinetics due to static effects on the distribution of emission probabilities within the initial ensemble of chromophore-solvent configurations. The dependence on Aem should have a dynamic origin and could mean higher quenching probabilities of the relaxed excited state species by the solvent environment. In Table 2, the results for indole in isobutyl alcohol are presented at different temperatures. They witness for the absence of any significant emission wavelengthdependent variations of the "pure" emission decay component whether the excitation is performed at the maximum of absorption or at the red edge, whatever the temperature. When the excitation is performed at the red edge of the absorption spectrum, however, the measured lifetime can be either longer or shorter than the lifetime obtained after excitation at the absorption maximum, depending on the temperature. At 20 "C, the excited state lifetime obtained after red edge excitation is significantly longer than after excitation at the maximum of absorption, whereas at -40 "C, it is significantly shorter (Table 2). The latter decrease of lifetime may be explained by photoselection of species with smaller energy separation between the ground and the excited state, which favors radiationless internal conversion to the ground state. This effect vanishes in the course of relaxation, which averages local environments. Time-Resolved Emission Spectra. TRES were reconstructed as described in Materials and Methods. One example of the set of spectra obtained in the first 5 ns of the fluorescence decay at -40 "C is represented on Figure 6. The variation of the barycenter in frequency as a function of time shows a steep decrease from VO values comprised between 29 830 cm-l at 20 "C and 30 396 cm-I at -40 "C toward a similar plateau value for the different temperatures (90= 29 100 cm-') (Figure 7a)
time
wavelength (nm)
Figure 6. Three-dimensional representation of a "RES experiment. Indole in isobutyl alcohol at -40 "C. Each emission spectrum is reconstructed by steps of 100 ps.
except for the curve at 20 "C which exhibits a higher plateau value than the other decays at lower temperatures. The apparent decrease in PO value with temperature can be due to a limited resolution of the instrumentation both in time (20 ps time resolution) and wavelength (5 nm bandwidth). A maximum shift of -1300 cm-' is therefore observed at the lowest temperature, corresponding to a stabilization energy of -3.7 kcaVmol. This spectral shift is less important than the expected one of 1800 cm-' observed by cooling alcoholic indole solution down to 113 K, which corresponds to a stabilization energy of 4.8 kcaVm01.~~~ This difference can be due to the limited time resolution which does not allow to detect processes faster than -20 ps. The C(t) function (Figure 7b) was calculated from the barycenter data and was fitted to a "quasi infinite" set of exponential components (1 00 equally spaced in log z scale) with a MEM program. The results show that three major discrete species are revealed in the C(t) decay at low temperatures (a minor fourth component may be present at -40 "C) and only two at higher temperatures, the fastest decay time becoming too short to be measurable (Figure 8). In no cases have we found broad continuous distribution of rates. The longest decay time values at different temperatures are in good agreement with the longest longitudinal relaxation time values measured by dielectric method in the same temperature range!(). It is to be remarked that no longer decay times than the longitudinal dielectric relaxation time can be observed but only the shortest ones. The fast decay time evidenced by analysis of the decay curves obtained in the blue and red edges of the fluorescence emission spectrum displays intermediate values as compared to the decay times revealed by the TRES analysis. The Arrhenius representations for the two longest time components are linear (Figure 5) and display similar slope providing an activation energy of 7.8 f 0.3 kcaVmol for both time components. This estimation is in good agreement with that obtained from the variation with temperature of the short component in the analysis of single decay curves in the blue and red regions of the fluorescence emission spectrum and in good agreement with the activation energy of the relaxation process measured in isobutyl alcohol in the same temperature range!0h This suggests that both approaches follow the same physical processes.
Solvent Relaxation around the Excited State of Indole
J. Phys. Chem., Vol. 99, No. 41, 1995 14937
30.2 30.4'-
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Figure 7. TRES data of indole in isobutyl alcohol. (a) evolution with time of the barycenter at different temperatures; (b) In [C(t)]representation; (c) variation of the width at half-maximum as a function of time at different temperatures.
There is no detectable change in shape of the spectra as a function of time in the explored time range. The half width of the spectrum is increasing by -380-400 cm-l The major part of the effect occurs in a time range faster than the barycenter shift. At 20 OC, most of the effect occurs probably in a much faster time scale, almost undectable with our instrumentation (Figure 7c). The higher plateau value of the barycenter at 20 "C with respect to lower temperatures can be therefore due to the averaging of an inhomogeneous spectral broadening occurring at low temperatures as already n~ticed.~'
Discussion Lifetime Distributions and Spectral Shifts: Complementarity of Approaches. Electronic excitation results in redistribution of chromophore electronic density and in changes of dipole moment (which usually increases). This modifies the interactions with the solvent molecules which are at equilibrium in the ground state and become out of equilibrium at the moment of excitation. The relaxation toward the thermodynamic equilibrium consists mainly in rotational equilibration of the chromophore and of solvent dipoles, but also in translational
0.1 1 time (ns)
10
Figure 8. Distributions of relaxation times obtained by MEM analysis of C(t). (A) -40 "C (TI = 4.01 ns; t 2 = 1.06 ns; r3 = 0.42 ns; t4 = 0.14 ns; al= 434 cm-I; a2= 482 cm-l; a3= 48 cm-l; Q = 247 cm-I); (B) -20 "C (TI = 0.95 ns; r2 = 0.42 ns; t3 = 0.15 ns; a1 = 170 cm-l; a2 = 348 cm-I; a3 = 230 cm-I); (C) 1 "C ( t l= 0.29 ns; t 2 =0.08 ns;; aI= 289 cm-l; a 2 = 456 cm-l) and (D) 20 O C ( t l= 0.95 ns; r2 = 0.42 nss; aI= 295 cm-'; a2 = 254 cm-I).
motion.'-3 It reduces the energy level of the excited state and, thus, shifts the fluorescence spectrum toward lower energies. The relaxation is superimposed on the fluorescence emission kinetics and results in a complex dependence of emission intensity on energy and time, I(v,t). The problem of describing this process is further complicated by the fact that, at time zero, the emission is distributed in energy described by a spectrum Io(Y), which contains both homogeneous and inhomogeneous broadening components. The former is determined by intrinsic properties of the chromophore and its solvent cage and the latter reflects the distribution of solvent cages which differ in interaction energies. Therefore, the relaxation results not only in decrease of the average energy but also in time evolution of the emission contour. The spectroscopic experiments with high time resolution allow us to obtain I(v,t) and our goals are (i) to characterize the solvent dipolar relaxation and to distinguish this process from others which may result in changes of the energy of emitted quanta (intramolecular chromophore isomerizations, proton and electron transfer, formation of exciplexes, etc.) and (ii) to describe this process quantitatively in terms of kinetic constants or relaxation times. The common procedure in this analysis is to consider series of functions, depending on one parameter, emission energy (or wavelength), or time at fixed values of the others. The construction of Zi(t) curves at different Vi over the emission spectrum is referred to as single-decay analysis and the
Vincent et al.
14938 J. Phys. Chem., Vol. 99, No. 41, 1995 construction of Ij(v) curves, which are the spectra at different time values, tj, is referred to as reconstruction of time-resolved spectra. Both these complementary approaches have their advantages and limitations. The single decay curves are derived directly from the experiment. In the case of dipolar relaxations (as well as in the case of any excited state reactions) they may be deconvolved to a minimum of two functions, one describing the emission decay Z’(t) and the other the relaxations, Z”(t). While the emission decay function may be usually considered singleexponential, Z’(t) = exp( -t/ZF), the relaxational decay function Z”(t) may be very complex. It should describe the temporal depopulation of excited chromophores, emitting at high energies as well as the temporal repopulation of chromophores with low energies of emission. Therefore, its sign should be positive or negative, respectively, in the short- and long-wavelength range. It may not be described by single-exponential function, since the kinetics may be inhomogeneous, due to the possible existence of a distribution of relaxational rates in the ensemble of the solvation shells. This function should even not be expected to be unimodal a priori, since a number of solutesolvent modes may participate to the relaxation. Deconvolution of complex function into a number of single-exponential functions presents a serious problem. This is the advantage of MEM analysis to handle a large set of parameters with the least initial guess and to sort out time constant distributions distinguishable from discrete component^.^^-^*^^^ In contrast to single-curve decay analysis, the construction of TRES exhibits the advantage that the full contour of the emission spectrum is simultaneously included into the analytical procedure and leads to visually demonstrative results. However, the procedure of obtaining and analysis of TRES requires several steps, which may be the source of systematic errors and artifacts. The TRES analysis involves three separate steps of nonlinear regression which are the fitting of the emission transients at each wavelength to a multiexponential form, a fitting the instantaneous spectrum to an analytical (usually log-normal) function and a fitting of C(t) to a multiexponential form.34The presented time-resolved spectra are usually normalized to equal intensity (spectrum surface or height at the maximum emission wavelength), so the information on possible coupling of spectral motion with the emission decay is lost. The C(t) correlation function is sensitive to the assignment of C(0) and C(=)values to eq 8,35,36and if the spectra change their shapes in the course of relaxation, to the manner by which v(t) is chosen (barycenter or band maximum).36 Our results demonstrate that the transition from the decay kinetics space to the lifetime distributions space with the application of MEM offers new possibilities in the study of dynamics of molecular relaxations. 1. Analysis of multicomponent decays is performed without any restrictions on their number, position and sign. This allows in particular to single out the Z’(t) function and study its dependence on emission energy. This could resolve the cases of inhomogeneous emission decay kinetics, which may be due either to the inhomogeneous distribution of decay rates in the initial ensemble of solvates or to the effects of quenching, associated with the relaxation. The peculiarity of indole is that its fluorescence emission kinetics does not exhibit strong inhomogeneous behavior, being not significantely different at the short- and long-wavelength sides of the emission band at different temperatures. Moreover, as we observed, it changes only to a slight extent at the red-edge excitations. The problem of participation of inhomogeneous decay kinetics in the relaxation process has been extensively discussed in the
literature$’-43 MEM analysis of emission decays offers a direct solution of this problem. 2. MEM analysis allows to resolve negative components in lifetime distribution. This is especially important for the observation of temporal increase of the number of low-energy emitters, which should result in the negative component(s) in the range of long emission wavelengths. In our experiments the negative component is clearly observed as a mirror image of the positive component, existing at shorter wavelengths. As expected, it follows the same regularity as the positive component in terms of dependence on solvent and temperature. It should be noted that if the data are processed into TRES analysis, the negative component may be undetected. The time shift of the TRES may be observed even in the case when negative components are absent. In these cases, the motions of the spectra originate from another mechanism, inhomqgeneous kinetics of emission decay,’”-43 but also quenching, if the relaxation itself is associated with a decrease of the ZF value. The TRES are extremely sensitive to the change of ZF in the course of relaxation, while their analysis does not provide the intrinsic test for these effects. For some fluorescence probes ZF is strongly solvent viscosity dependent, which in the case of distributed relaxations may result in different lifetimes for unrelaxed, temporarily rigid environments and for relaxed, temporarily liquid ones.44 Single wavelength measurements and analysis are important in this respect. 3. MEM analysis of lifetime distributions allows to test the important assumption, inherent in most theories in spectroscopy of molecular relaxations, that the emission decay function Z(v,t) is the product of two independent contributions, one describing the time-dependent light emission and the other describing the spectral relaxation in terms of spectral shift:
Z(v,t) = Z&{v
- v(t)} exp(-t/tF)
(8)
+
with v(t) = v, A v exp(-t/zR) and A v = vo - v. Thus, in the simplest case of a single excited state lifetime z, and a single relaxation time ZR, the decay and spectral changes in time are uniquely characterized by ZF, an elementary spectral contour of invariant shape and a spectral position parameter, v(t), which decays exponentially with the relaxation time ZR. The decay law at the emission frequency v may be presented as follows after serial expansion:
+
Z(v,t) = Z0[@{V - v,} exp(-t/z,) Fl(V)exp(-t/rR - t k F ) F z ( Y ) exp(-?/rR - 2t/rF)] (9)
+
Retention of the fist terms gives
Z(v,t) = Zo[e{v - v,} exp(-t/z,)
+
F l ( Y ) exp(-tlz,
- t / r 4 (10)
Thus, within this approximation, the position of the experimental value of the relaxational component in the emission decay zrel allows the determination of relaxation time ZR: 1/zre1= l/Z,
+ l/ZF
However, in some cases, this approximation is not fully valid since the contribution of the higher terms can far exceed the contribution of the first term. This can occur for instance at the blue edge of the fluorescence emission spectrum. In this case, the peak in the decay time distribution that corresponds to the TDFS can be shifted to shorter values.43b In the case of a single relaxational process, a single rate of spectral relaxation characterizes the whole envelope of the
J. Phys. Chem., Vol. 99, No. 41, 1995 14939
Solvent Relaxation around the Excited State of Indole emission spectrum. It should be mentioned, however, that the presence of positive and negative components with their absolute values independent on the position in the emission spectrum is also a characteristics for two-state reactions (e.g., formation of excimers or exciplexes). In these cases, the TRES should describe the disappearance in time of one component and appearance of the other. At intermediate times, it should contain the contribution of the two components, which can be detected by a substantially increased width of the spectrum. In contrast, in the cases of solvent relaxation, the spectral width can increase due to the existence of a distribution of solvent cages with different interaction energies in the relaxed states but should level up to an asymptotic value. The results of the studies on indole in isobutyl alcohol show that it is the case. The maximum increases in spectral width levels up at about 360 cm-’. 4. MEM analysis of the C(t)function allows to discriminate between distributed relaxation and discrete processes.3ob Our results show that the latter situation prevails and therefore several coupled or independent relaxation processes differing in their rates are occurring around the excited solute. 5. The MEM analysis of lifetime distributions allows easy, singular, and visually representative analysis of red-edge effects in molecular relaxations. The site-selective excitation at the red edge should excite only those chromophores within the distribution, the environment of which is very close to the relaxed state. Thus, the temporal depopulation at short emission wavelengths and repopulation at long wavelengths should be suppressed. This should result in the disappearance of the “relaxational” component in the lifetime distribution. In timeresolved spectra this effect is manifested as the disappearance of motion in time of the spectrum, which occupies the strongly shifted long-wavelength position.I7 Such a shift, however, and the absence of time-dependent mobility do not allow to draw a unique interpretation and in some cases the presence of impurities is suspected. In lifetime distributions, in contrast, the ZF peak, which is obtained in the same experiment and occupies the same position, is a strong argument against such interpretations. The invariance of ZF at different ,lex is one of the assumptions for our method of estimation of ZR from red-edge effects in the steady-state ~ p e c t r a . ’ ~ .In ’ ~this - ~ ~case the time-integrated form of C(v,t) is used with the assumption that C(v,O)depends and C(v,-) does not depend on ,lex. Application of the BakhshievMazurenko model’ allows to obtain a simple correlation between ZR and ZF:
+ ZF)
C(v)= (v - vedge)/(vo- vgedge) = t,/(tR
(12)
Here v and vedgerefer to the position of fluorescence spectra at the mean and red-edge excitations, and the subscript 0 indicates that the spectra should be taken at t = 0 or in rigid environment (at low temperatures). This method is of value in the studies of proteins, where ZR are long and variations of solvent and temperature in order to reach Y, are very limited. As known, the red-edge effects may be both static and dynamic. The static effect allows producing site photoselection within stationary inhomogeneously broadened absorption band, which occurs in low-temperature solvent glasses. The dynamic effect is concerned in site-photoselection at the first instant of relaxation. As the relaxation proceeds, the redistribution of environments with different energies occurs as a function of time with a loss of correlation in time. Thus, the properties of the species excited at the maximum and at the red edge, being different at the time of excitation, level out in the course of relaxation. Thus, the dynamic red-edge effect, which in our
case is represented by the disappearance of the relaxational component in the lifetime distribution, may be considered as a proof for solvent dielectric relaxations and for the excited state reactions, which are coupled with them directly. The timedependent shift of spectra for different excited state reactions may occur on a time scale much slower than the dipolar relaxations. In this case the local chromophore-solvent configurations may be averaged on a much faster time scale and no red-edge effects should be observed.46 Photophysics of Indole in Polar Environments: The Importance of Solvent Relaxation. Indole, as the side chain of tryptophan, is responsible for most of the absorption and fluorescence properties of proteins, which allows to use it as a natural spectroscopic probe in the studies of protein structure and dynamics.’* The popularity of fluorescence studies of proteins is mainly due to the fact that the parent indole molecule exhibits extreme sensitivity of fluorescence spectra to its environment, which indicate high polarity of the excited stateIs and probably, the ability to participate in specific b ~ n d i n g . ~ ~ - ~ ~ Depending on the solvent, the emission maxima of these spectra vary within a wide range between 28 200 and 32 800 cm-’. Thus, between hexane and water, the spectral shift is as much as 4600 cm-’ ?’ Very significant in glass-fonning polar solvents are the temperature-dependent shifts-by 3000 cm-’ or more-for the transition from liquid solvent to low-temperature g l a ~ s . ~ ’ - ~ ~ Such a strong dependence on temperature may be the result of several photophysical events, which include dipolar relaxation but are not limited to relaxation. Substantial polar interactions should result in broad distribution of interaction energy and inhomogeneous broadening of spectra. This leads to easy observation of red-edge effects, which are based on photoselection of indole environments with the strongest excited state interactions.”-’3,’s These effects are observed in rigid environments and, in glycerol at 77 K, the shift of fluorescence spectra upon increase of excitation wavelength, may be as large as 2630 cm-’ (from 307 to 334 nm).46 This effect decreases as a function of increasing relaxation rate and vanishes in highly mobile liquid environments. The excitation wavelength of 300 nm is the point at which the steep dependence of fluorescence maximum on excitation wavelengths in solid polar solution (glycerol at 77 K) almost crosses the Lex independent function in liquid environment. At this point, the relaxation is not associated with change in the mean excited state energy. The present studies of lifetime distributions demonstrate the complete disappearance of relaxational components at this excitation wavelength. Previous applications of time-resolved spectroscopy to indole and its derivatives, including tryptophan, allowed to observe time-dependent events in the range of solvent relaxation^,^^-^^ but were not conducted in sufficient detail to resolve the question as to the origin of the time-dependent spectral shifts: general solvent effects (orientationalrelaxations of dipoles) or formation of specific complexes.53b The impossibility for our data to evidence more than one single ZF values with small variations as a function of solvent and temperature is evidence for the absence of excited state reaction beside solvent relaxation, from which should originate the large Stokes shift observed in TRES experiments. The existence of a red-edge effect is also in line with this conclusion. The frequently discussed formation of solute-solvent excited state complexes (exciplexes)I2 is not supported by the present results. The exciplex model of spectral shifts may be in correspondence with our data only at two conditions: (i) the normal and exciplexed forms should have essentially the same lifetime ZF and (ii) the red-edge excitation at 300 nm should
14940 J. Phys. Chem., Vol. 99, No. 41, 1995
produce an ideal selection of an exciplex state already existing before the relaxation. In our view, a variety of experimental data may be fit to models in which the exciplexes preexist as ground-state complexes or are formed at a very short time before the relaxation. This complexation would stabilize the large excited state dipole moment of indole and the dipolar relaxation occurs relative to this complex. These ground state comare subjected to small and very fast rearrangements upon excitation. This exciplex attains a large dipole moment of 12- 14 D,57 which is responsible for large effects of solvent dipolar relaxations on fluorescence spectra. The observation of a dynamic red-edge effect is more consistent with the solvent dipolar relaxation mechanism, rather than with specific complex formation. Whether a single or several relaxation modes are present is not resolved by the single emission wavelength data since peaks broader than obtained by simulated data are recovered. In contrast, TRES experiments have allowed to separate the contribution of the fluorescence decay from that of the spectral shift and to recover discrete relaxation time constants. The C(t) functions (defined by the barycenter of the emission spectrum) show in fact an obvious feature whatever the temperature: it cannot be analyzed as a single exponential decay. This was observed extensively by other authors for different probes and solvents.36 At low temperatures, three discrete relaxation times are derived by MEM analysis of C(t)as a series of exponentials. The fastest one becomes in fact soon at the limit of the detection and is not measurable anymore at temperatures higher than comprised between -20 and 1 “C. It is likely that the long time components are meaningful and describe different relaxation processes in the indole solvent shell. The longest relaxation decay values measured at different temperatures fit remarkably well with the long longitudinal relaxation time values T Iobtained by dielectric measurements in a much wider temperature range.‘”’ The intermediate relaxation time values fit to a poorer extent with the second relaxation time z2 but this can be due to the extremely different temperature ranges of these two studies. z2 was measurable separately from T I only at very low temperatures by dielectric dispersion measurements which were not explored yet in the present fluorescence measurements. The fastest decay time could represent an extremely fast reorganization of few .solvent molecules in the solvent cage. The indole ring can in fact disturb the local solvent structure by being both H-bond donor or acceptor. Lower temperature experiments with the TRES technique as well as studies with shorter alcohols are needed to settle this point.
Conclusions In conclusion, we have demonstrated that the emission decay kinetics of indole in polar protic solvents can be described by MEM analysis in terms of model-free lifetime distributions. These distributions consist of two discrete exponential components only and not of a broad distribution: a first discrete longlived decay and a second one always observed at shorter times. The study of the dependence of these components upon excitation and emission wavelengths in different solvents and at different temperatures allows to ascribe the discrete longlived component to the emission decay, while the short-living component is the result of solvent molecular relaxations occurring during the time of emission or earlier. Comparison of these observations with the time-resolved spectra, generated on the same basis of primary data, allows to perform comparative critical analysis of the two techniques and demonstrates their complementarity. The MEM analysis of decays is suggested to be the primary test, which should determine the choice
Vincent et al. of the model for further data analysis for adequate description of the photophysical process. For indole in polar solvents no indication of excited state reactions other than the dipolar relaxations was found in the time range longer than -20 ps. The emission decay component is represented by a single narrow peak in lifetime distribution and exhibits very small dependence on solvent and temperature. No dependence on emission wavelengths can be observed but a slight change with excitation wavelength. The relaxational component exhibits distributed character and strong dependence on solvent and temperature, in line with the data on solvent relaxation rates in these conditions. TRES measurements and analysis by MEM show that the solvent relaxation process cannot be described either by a continuous distribution or by a single time constant. An interesting observation is that no relaxation time longer than the long longitudinal relaxation time determined by dielectric measurements can be evidenced but only shorter ones.4o While the first observation agrees with a “continuum” model of solvent which applies in cases of solvent molecules in weaker interactions with the probe than with their partners and therefore a collective reorientation of the solvent molecules in the solvation shell, the second observation concerning shorter processes is not included in this model. This fast component can be due either to single molecule rotational motion, to OH dipole relaxation, or to fast equilibration of H-bonding, but further studies at lower temperatures and as a function of the chemical nature of the solvent should be performed. Whether the dipolar relaxation phenomenon evidenced in the protic solvent can explain fast excited state reactions detected recently in proteins must await further
investigation^.^^ Acknowledgment. The technical assistance of S. Tosti is gratefully appreciated. The technical staff of LURE is gratefully acknowledged for running the synchrotron ring during the beam sessions. The helpful comments from Dr. E. P. Petrov from the Institute of Physics (Academy of Sciences of Byelorussia, Minsk) are appreciated. A.D. acknowledges a visiting professorship grant from the French Ministkre de 1’Enseignement Suptrieur et de la Recherche. M.V. acknowledges the Institut National de la Sante et de la Recherche Medicale for its continual financial support. References and Notes (1) (a) Mazurenko, Y. T.; Bakhshiev, N. G. Opt. Spectrosc. 1970, 28, 490. (b) Bakhshiev, N. G. Spectroscopy of intermolecular interactions. Nauka: Leningrad, 1982. (2) Maroncelli, M.; MacInnis, J.; Fleming, G. R. Science 1989, 243, 1674. (3) (a) Barbara, P.; Walker, G. C.; Smith, T. P. Science 1992, 256, 975. (b) Simon, J. D. Acc. Chem. Res. 1988, 21, 128. (4) (a) Pavlovich, V. S.; Pikulik, L. G. Isu. Acad. Sci. USSR, Ser. Phys. 1975, 39, 2373. (b) Palovich, V. S.; Petrushkevich, P. P.; Pikulik, L. G. Opt. Spectrosc. 1979, 46, 898. ( 5 ) Chakrabardii, W. R.; Ware, W. R. J. Chem. Phys. 1971,55, 5494. ( 6 ) Ware, W. R.; Lee, S . K.; Brant, G. J.; Show, P. J. Chem. Phys. 1971, 54, 4729. (7) DeToma, R. P.; Easter, J. H.; Brand, L. J. Am. Chem. SOC.1976, 98, 5001. (8) Bagchi, B.; Oxtoby, D. W.; Fleming. Chem. Phys. 1984, 86, 257. (9) Mazurenko, Yu. T.; Udaltsov, V. S. Opt. Spectrosc. 1978,44,417. (10) (a) DeclCmy, A.; Rullibre, C.; Cottis, Ph. Chem. Phys. Lett. 1987, 133, 448. (b) DeclCmy, A.; RulliBre, C.: Kottis, Ph. Laser Chem. 1990, 10, 413. (c) Declkmy, A.; Rullibre, C. in Ultrafast Reaction Dynamics and Soluent Effects; Gauduel, Y . , Rossky, P. J., Eds.; AIP Press: New York, 1994, Vol. 298. (c) Horn, M. L., Gardecki, J.; Papazyan, A.; Maroncelli, M. Subpicosecond measurements of polar solvation dynamics: coumarin 153 revisited, submited to J. Phys. Chem.
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