The Journal of
PhysicaIChemistry
0 Copyright 1994 by the American Chemical Society
VOLUME 98, NUMBER 41, OCTOBER 13,1994
FEATURE ARTICLE Vibronic Spectroscopy of Individual Molecules in Solids Anne B. Myers,*JPaul TchCnio,' Marek Z. Zgierski3 and W. E. Moerner*J IBM Almaden Research Center, 650 Harry Road, K95/801, San Jose, Califomia 95120-6099; Department of Chemistry, University of Rochester, Rochester, New York 14627-0216;and Steacie Institute for Molecular Sciences, National Research Council, Ottawa, Canada KIA OR6 Received: April 22, 1994; In Final Form: July 6, 1994@
Spectroscopic selection and detection of individual molecules in the solid state provide a means of eliminating the inhomogeneous broadening that is a ubiquitous feature of condensed phase molecular spectra. Over the past few years the feasibility and utility of several types of electronic spectroscopy have been demonstrated at the single-molecule level. This article briefly summarizes that work and describes our recent extensions into the realm of single-molecule vibrational spectroscopy. This promising technique should allow much more detailed probing of the local environment of individual chromophores, particularly in highly disordered solids such as polymers and glasses.
1. Introduction to Single-Molecule Spectroscopy A. Rationale. Nearly all types of spectroscopy involve detection of a signal that has contributions from a large number of different molecules. It is often possible to detect the result of a single molecule interacting with a radiation field-fluorescence spectroscopy, for example, typically utilizes single photon counting detection, with each detected photon coming from a single molecule-but the detection of one photon is not very interesting. In order to obtain the statistics needed to do meaningful spectroscopy many photons are required, and they usually come from many different molecules. This would not matter if all the molecules were identical, but in practice they never are. Even in a low-pressure vapor consisting of a single chemical species there is a distribution of translational, rotational, and often vibrational states, and the fact that different initial states give rise to different spectra can severely comprot University of Rochester.
Current address: Laboratoire A. Cotton, CNRS II, Universit6 Paris XI, Bat. 505, 91405 Orsay, Cedex, France. 5 National Research Council. II IBM Almaden. Abstract published in Advance ACS Absrracrs, September 1, 1994. @
0022-365419412098-10377$04.50/0
mise the ability to perform high-resolution spectroscopy. It is for this reason that spectroscopy in supersonic molecular beams, where the distribution of initial quantum states is greatly narrowed, has become such a powerful and popular technique. In condensed media, each molecule has slightly different potential energy functions due to a slightly different arrangement of the other molecules in its local environment. Even at low temperatures where the thermal distribution of initial quantum states for each molecule is quite narrow, inhomogeneous broadening of spectra due to this distribution of environments is significant, even in apparently well-ordered crystals and particularly in amorphous media such as glasses, polymers, and proteins. A number of creative experimental techniques have been developed to attempt to remove andor quantitate inhomogeneous broadening phenomena, particularly in low-temperature solids. These include time-resolved techniques such as the photon and the frequency-domain spectroscopic techniques of spectral hole b ~ m i n g , * * ~emission - ~ - ' ~ (fluorescence1 phosphorescence) line n a r r o ~ i n g , ~ . ' ~ and , ' ~ -line-narrowed ~~ four-wave m i ~ i n g . ~The ~ - latter ~ ~ techniques exploit the fact that a single molecule's spectrum is much narrower than that 0 1994 American Chemical Society
10378 J. Phys. Chem., Vol. 98, No. 41, 1994 of the ensemble, so excitation with a narrow-bandwidth laser can select out of the inhomogeneous distribution a subset of molecules absorbing within a narrow frequency range. These methods have enjoyed considerable success in allowing relatively high resolution spectroscopy to be performed in the presence of strong inhomogeneous broadening and in revealing aspects of the physics of the inhomogeneous broadening process itself. However, it remains abundantly clear that these energyselective techniques do not entirely remove inhomogeneities; that is, the subset of molecules that happen to have a particular spectroscopic transition at the same energy may still be very different in other respects. The ultimate in removal of inhomogeneous broadening is to perform spectroscopy on one molecule at a time. This not only removes spectral congestion due to ensemble averaging effects but also, by sequentially studying different single molecules within the ensemble, allows detailed probing of the nature of the different available environments. Over the past several years, three groups have demonstrated the feasibility of performing meaningful spectroscopic measurements on single chromophores in low-temperature solids, both mixed crystals and polymer g l a s s e ~ . ’ ~A~large ~ ~ -number ~~ of different types of spectroscopy have been demonstrated, and a rich variety of spectral and dynamic behaviors have been observed, some of which could not have been inferred from ordinary bulk spectroscopies or standard energy-selection line-narrowing methods. This article briefly reviews the experimental techniques, results, and prospects for this type of spectroscopy in general and then discusses in more detail the particular technique of vibrationally resolved fluorescence from single chromophores. It should be noted that there is presently a great deal of activity in the development of techniques for detecting fluorophore-labeled single macromolecules (particularly proteins and nucleic acids) in s o l ~ t i o n , ~as ~ - well ~ ~ as imaging single fluorophores using near-field scanning optical microscopies under room-temperature condition^.^^-^^ These techniques promise many exciting future applications, but room-temperature fluorescence spectra, even from single molecules, are expected to be too broad to provide the kind of detailed vibrational information that is the subject of this paper. B. General Experimental Approach. As one contemplates performing a spectroscopic measurement on a single molecule, two fundamental questions present themselves. First, will the signal from one molecule be large enough to detect, both in an absolute sense and relative to whatever background may be present? Second, how can a single molecule be isolated for study? The answer to the first question depends on both the type of spectroscopic measurement to be performed and the photophysics of the molecule of interest. As an example, we consider the most common type of single-molecule spectroscopy, that of fluorescence excitation, where the excitation frequency is scanned while detecting “total” fluorescence as a monitor of absorption. The large aromatic hydrocarbon molecules on which all of the studies to date have been performed (pentacene, perylene, and tenylene; see Figure 1), excited in the region of the S 1 SO origin transition, can be considered for the purpose of estimating fluorescence emission rates as three-level systems: the zero-point levels of SO and of S1 and a triplet which acts as a “dark” bottleneck state. Using the simple kinetic scheme of Figure 2, one can derive the following expression for the maximum fluorescence emission rate at a saturating incident light intensity, R_:30
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Myers et al.
fyJ 0 0
Figure 1. Structures of the single-molecule probes discussed: (a) pentacene, (b) perylene, and (c) tenylene.
1 (So)
I
L
ll
-
Figure 2. Three-level system used to describe saturation behavior of the SI SO fluorescence of aromatic hydrocarbons.
where k21, k23, and k31 are the rate constants indicated in Figure 2 and q 5 ~is the fluorescence yield. Inserting the appropriate photophysical parameters for the most widely studied single molecule system of pentacene in p-terphenyl near 1.5 K yields a calculated maximum emission rate of 3 x lo6 photons s-l, an enormous signal!30 This, however, assumes that every emitted photon is detected, and it does not address the presence of background signal due to dark counts, scattered laser light, Raman scattering from the matrix, etc. A more relevant parameter is the signal-to-noise ratio, which can be approximated as36
rE
Dq5,oPtlAhv
SNR =
Ahv
1/2
+ C,Pt + N d t ]
(2)
Here D is the overall efficiency for detecting emitted photons, P is the incident laser power, t is the integration time, A is the area of the focused beam, hv is the incident photon energy, Nd is the dark count rate, and cb is the “light” background count rate per watt of excitation power. The peak absorption cross section, 0, saturates with increasing laser power according to CJ = ad(1
+ Z/ZS)
(3)
where 00 is the low-intensity limiting cross section and Is is the saturation intensity defined in ref 13. A large peak absorption cross section is important for maximizing the signalto-noise ratio. Broadening of the absorption line, with a corresponding reduction in the peak cross section, does not necessarily reduce the maximum count rate since the higher saturation intensity allows higher laser powers to be used, but the background count rate also increases with increasing power, reducing the signal-to-noise ratio. The dependence of the signalto-noise ratio on the experimentally variable parameters for the pentacenelp-terphenyl system is discussed in detail in ref 37. It is found both theoretically and experimentally that SNRs on the order of 20 in a 1 Hz bandwidth can be achieved quite readily, although this depends strongly on the light background
Feature Article which can vary considerably among different samples and experimental configurations. Thus one concludes that if the physical system and the experimental setup are properly chosen, the fluorescence from a single molecule can be quite strong enough to be detected with a decent signal-to-noise ratio. The isolation of a single molecule for study can be achieved by making use of scanning or imaging techniques in very dilute samples.47,56-58,61 Alternatively (or additionally), one can take advantage of the fact that in relatively rigid aromatic molecules at very low temperatures the homogeneous line width of the zero-phonon electronic origin transition is normally many orders of magnitude narrower than the full inhomogeneous width. Thus if the laser bandwidth is made narrower than the homogeneous origin width, at any chosen excitation frequency within the inhomogeneous band only a small fraction of the total number of molecules within the excitation volume are actually on resonance with the laser, and as the laser is tuned farther away from the peak of the absorption band, eventually the density of absorbers in frequency space becomes small enough that at any chosen laser frequency there is, at most, one molecule on resonance. Generally the laser is focused to a small spot and the sample concentration is chosen such that the total number of chromophores in the illuminated volume is in the 103-104 range. Under these conditions one can be fairly certain that tuning the laser to the peak of the inhomogeneously broadened origin band will excite enough molecules to generate a fairly strong signal on which to align the optical system, yet singlemolecule features can be isolated without having to tune the excitation frequency excessively far into the wings of the distribution. The basic idea of isolating individual members of the inhomogeneous ensemble by their spectrally narrow absorption features is very similar to the physical basis for fluorescence line narrowing and persistent spectral hole burning spectroscopies, but those techniques typically use considerably more concentrated samples and/or larger illuminated volumes such that there are always a fairly large number of molecules on resonance. It should also be noted that successful singlemolecule spectroscopy requires a chromophore having extremely low quantum yields for photochemical or photophysical hole burning. This can be appreciated quickly by realizing that a very good fluorescence detection efficiency (D in eq 2) is on the order of 0.01, and observation of a feature in a fluorescence excitation scan generally requires detection of at least lo3 photons, so a molecule simply will not be observed unless its zero-phonon absorption line remains at the same frequency long enough for it to emit at least lo5 fluorescence photons. This means that its hole burning quantum yield cannot exceed and must be orders of magnitude smaller if any detailed spectroscopy, such as vibrationally resolved emission, is to be performed. The need for extremely low hole burning efficiencies is probably the most stringent requirement on potential guesthost combinations for single molecule spectroscopy. To date, all of the experiments of the type described in this article have been performed at very low temperatures, below about 10 K and mostly near 1.5 K. These experiments become increasingly difficult at higher temperatures because pure dephasing processes rapidly increase the “homogeneous” electronic line width above its lifetime-limited value, reducing the peak absorption cross section 00 which is the critical molecular parameter in eq 2. Spectral diffusion and some hole burning mechanisms also become increasingly facile with increasing temperature. C. Single-Molecule Spectroscopy in Mixed Crystals: Pentacendp-Terphenyl. Detection of single-molecule features
J. Phys. Chem., Vol. 98, No. 41, 1994 10379 by frequency selection within an inhomogeneous ensemble was first achieved in the mixed crystal system of pentacene in p - t e r p h e n ~ l . The ~ ~ low-temperature spectroscopy and photophysics of this system had been characterized quite thoroughly by a wide variety of bulk spectroscopic techniques,1*62-64 and it was known to possess many of the properties desirable for single-molecule detection: very high photochemical and photophysical stability, a fairly strong SI SOorigin transition, a relatively low intersystem crossing yield and a short triplet state lifetime, a high fluorescence quantum yield, and a convenient absorption wavelength (near 593 nm). Pentacene molecules occupy one of four distinct sites in the p-terphenyl matrix whose origin transitions are denoted 0 1 - 0 4 ; single-molecule experiments have focused on the two reddest ones, 01 and to a lesser extent 0 2 . The inhomogeneous widths of these transitions are highly dependent on sample growth and handling but are typically in the few gigahertz range, whereas the lifetime-limited origin line width is only 8 MHz. The f i s t step toward single-molecule detection was the observation of statistical fine structure, the “bumpiness” in the absorption spectrum due to the presence of a small but finite number of absorber^.^^,^ The experiments of ref 25 then employed a double modulation technique (frequency modulation (FM) spectroscopy combined with Stark shifting) to detect single molecules in direct absorption. These experiments achieved a signal-to-noise ratio of only about 1, consistent with a subsequent analysis of the signal-to-noise limits in FM detection.37 A year later, Omt et al. demonstrated that fluorescence excitation rather than direct absorption allowed greatly improved signal-to-noise ratios to be obtained,28and virtually all subsequent single-molecule experiments on this and other systems have employed detected fluorescence as the method to measure absorption. Pentacenelp-terphenyl has become the “hydrogen atom” of single molecule spectroscopy-the system on which virtually every type of single-molecule measurement is f i s t made. Experiments reported thus far include studies of the origin line width31 and its temperature dependence?O saturation behavior,30 the observation of spectral d i f f u s i ~ n ,direct ~ ~ time-domain lifetime measurement^,^^ polarization Stark effect studies,34 pressure effects$5 the use of photon correlation techniques to study both photon bunching28,44and antibunching,35 optically detected magnetic r e s o n a n ~ e , “ and ~,~~ fluorescence imaging.50 D. Single-Molecule Spectroscopy in Doped Polymers: PeryleneRolyethylene and TerrylenePolyethylene. While pentacene in p-terphenyl does exhibit an inhomogeneous origin line width that is orders of magnitude larger than its homogeneous width, the extent of inhomogeneity is still rather small in this mixed crystal system. The variation in available environments is far greater in amorphous solids, which typically exhibit low-temperature inhomogeneous line widths exceeding 100 cm-’. The success of site-selective spectroscopic techniques such as fluorescence line narrowing and spectral hole burning for both obtaining high-resolution molecular spectra and exploring aspects of the matrix dynamics in low-temperature glasses prompted efforts to obtain single-molecule spectra in such environments. Since in spectral hole burning studies one often observed very narrow holes in p ~ l y e t h y l e n e , ~this *~~,~~ matrix was identified as a good candidate for single-molecule studies. Early efforts to detect single molecules of pentacene in polyethylene were unsuccessful, perhaps because of poor solubility leading to aggregation of the chromophore within the polymer. The f i s t successful single-molecule experiments in
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10380 J. Phys. Chem., Vol. 98, No. 41, 1994 polymeric glasses were performed on perylene in p~lyethylene.~~ Perylene is in many respects an even better chromophore for single-molecule studies than pentacene in that it has a stronger SI SO absorption and an even larger fluorescence quantum yield. Its main disadvantage is that its origin absorption lies in the blue at about 440 nm, a much less convenient wavelength for single-frequency dye lasers. While single-molecule features are clearly observable in perylene/polyethylene, the narrowest electronic origin line widths observed are at least a factor of 2 larger than the lifetime-limited This is assumed to arise from spectral diffusion processes occumng in the polymer at 1.5 K on a time scale faster than the several seconds required to scan the fluorescence excitation features. Spectral diffusion was also observed on longer time scales of seconds to minutes in the form of jumps between discrete resonance f r e q ~ e n c i e s . ~ ~ Finally, persistent spectral hole buming (light-induced changes in resonance frequency) was also observed in the polymer system. The hole-bumed molecules often returned spontaneously to the original frequency after a delay of 1-100 s, suggesting that the buming and recovery both involve rearrangement of only one conformational degree of freedom (twolevel system) of the matrix. More recently, a considerable amount of single-molecule work in polymers has been performed on the closely related system of terrylene in p ~ l y e t h y l e n e . ~Terrylene, ~ a higher homolog of perylene (see Figure l), also appears to have very desirable photophysical parameters, and its absorption maximum near 570 nm places it in a much more convenient spectral region. Terrylene in polyethylene exhibits spectral diffusion and hole burning qualitatively similar to that seen in perylene, but a wider variety of dynamics has been o b ~ e r v e d > ~Both - ~ ~ “random walking” and “state to state jumping” types of spectral diffusion occur$2 the power broadening and saturation vary considerably among molecules$2 and Stark effect studies imply large and highly variable local fields felt by molecules at different sites in the polymer.32 Unfortunately, unlike perylene, which has been studied extensively by a wide variety of bulk spectroscopic methods, the spectroscopy of terrylene is almost entirely uncharacterized. This proves to be a serious problem for interpretation of the single-molecule vibrational spectra of this system, as discussed in section 4. E. Single-MoleculeSpectroscopy in Shpol’skii Matrices: Terrylene in Hexadecane. Very recently, the first singlemolecule spectra in the polycrystalline environment of a Shpol’skii matrix have been reported for the system of terrylene in h e x a d e ~ a n e . ~The ~ , ~single-molecule ~ fluorescence excitation spectra in the origin region yield a low-intensity line width given approximately by the lifetime limit, as observed in the pentacenelp-terphenyl single crystal but in strong contrast with terrylene in polyethylene. Whereas single molecules of pentacene in p-terphenyl do not exhibit photoinduced hole buming, intensity-dependent electronic origin frequency jumps are seen in terrylenehexadecane, although the photophysical stability of terrylene in the Shpol’skii matrix appears to be considerably greater than in the polymeric environment. The ease of preparing Shpol’skii matrices compared with growing doped single crystals, combined with the likelihood of relatively high photophysical stability in such environments, makes these materials very attractive for future single-molecule studies.
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2. Environmental Effects on Vibrational and Vibronic Spectra of Aromatic Hydrocarbons Since the gist of the technique discussed in the remainder of this paper is the use of vibrationally resolved fluorescence spectra of single molecules to learn about their condensed phase
Myers et al.
TABLE 1: Ground-State Vibrational Frequencies of Perylene in Different Environments” isolated n-hexane PMMA pure moleculeb matrix‘ glassd crystal‘ 353 427 547
357
550
1300 1372 1580
717f 904 1298 1374 1578
355 43 1 549 709 903 1301 1369
550 1296 1373 1570
a All frequencies in cm-’. S1 origin excited fluorescence in free jet, ref 71; uncertainty 1 cm-’. Hg lamp excited fluorescence at 77 K, ref 72; uncertainty 10- 15 cm-I. Line narrowed fluorescence at 2 K, ref 23; uncertainty not given. ‘Fourier-transform Raman, room temperature, ref 73; resolution 3 cm-’. f Overtone transition.
TABLE 2: Ground-State Vibrational Frequencies of Anthracene in Different Environments” isolated argon n-heptane p-terphenyl pure moleculeb matrixC matrixd crystal‘ crystar 237 390 5 24 624 753 7788 912 1012 1100 1165 1183 1263 1382 1408
244 391,398 527 626 759 782-7979 924 1017 1176 1194 1275 1419
394
389-390
627 759 7898
623-625 753-757
1015
1007-1008
1163 1185 1257 1389 1407
1162-1163 1256-1258 1400-1403
242 395 5 19 622 753 90 1 1007 1103 1163 1187 1260 1376 1403
All frequencies in cm-’. SI origin excited fluorescence in free jet, ref 70; uncertainty 5 cm-I. Origin excited fluorescence at 12 K, ref 74; uncertainty ‘ 5 cm-’. Lamp excited fluorescence at 12 K, ref 20; uncertainty 1-5 cm-’. e Lamp excited fluorescence at 4 K, ref 75. Uncertainty not given; range of values encompasses emission frequencies from the five different origins in this environment. f Raman, room temperature, ref 76; uncertainty 1-2 cm-’. 8 Overtone transition.
environment and/or the nature of the interactions between the molecule and that environment, it is appropriate to briefly review the existing literature on environmental effects on ground-state vibrational frequencies and fluorescence intensities. We focus entirely on the polycyclic aromatic hydrocarbons that have been used successfully as single-molecule probes or are closely related to those that have. A. Effects on Ground-State Frequencies. High-resolution dispersed fluorescence spectra yielding ground-state vibrational frequencies for isolated molecules have been reported for a number of polycyclic aromatics by origin excitation in free jet expansion^.^^-'^ In addition, ground-state vibrational frequencies in a variety of environments are available from fluorescence and/or Raman s p e ~ t r a . ~ ~Table , ~ ~1, summarizes ~ ~ - ~ ~ data for perylene, one of the molecules used as a single-molecule probe. Tables 2 and 3 summarize vibrational data for anthracene and tetracene (naphthacene), the next two smaller analogs of the molecule most thoroughly studied by single-molecule techniques, pentacene. Only a few of the ground-state vibrational frequencies for pentacene itself in the vapor phase appear to have been published,80although vibrationally resolved spectra have been reported in a variety of mixed crystals and glassy solids.2,62,81 The data in these tables indicate that the ground-state vibrational frequencies of polycyclic aromatics do not, in general, depend strongly on environment. For the most part,
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TABLE 3: Ground-State Vibrational Frequencies of Tetracene in Different Environment9 isolated p-terphenyl naphthalene pure moleculeb host? hostd crystal‘ 307 612 738 986 1149 1177 1381 1431 1536 1615
311 616 758 996 1154 1178 1198 1385 1446 1546 1615
321 619 759 999 1162 1201 1388 1548 1621
317 620 754 lo00 1165 1183 1200 1387 1451 1547 1621
All frequencies in cm-’. S1 origin excited fluorescence in free jet, ref 69; uncertainty 10 cm-l. Lamp excited fluorescence at 4 K, ref 77; uncertainty not given. Lamp excited fluorescence at 20 K, ref 78; uncertainty not given. ‘Raman at 298 K, ref 79; uncertainty 2 cm-’.
the frequencies in pure crystals, mixed crystals, glasses, and isolated molecules do not vary by more than 10 cm-’. However, it is quite clear that in at least some cases the vibrational frequencies in different environments do differ by more than the uncertainty in the measurement. The former observation implies that only rarely will there be any question about the correspondence between vibrational transitions measured in different environments. The latter observation suggests that vibrational frequencies of polycyclic aromatics are at least weakly sensitive to their environment and may be of use as diagnostics of local environments in inhomogeneous media. B. Effects on Vibronic Intensities. The sensitivity of the relative fluorescence line intensities to the molecular environment is difficult to quantitate from the data available in the literature. Many fluorescence spectra are reported with only a qualitative mention of intensities, if that. Even when numerical intensity estimates are given, they tend not to be very accurate; they may correspond either to peak heights or to peak areas (not comparable unless the spectral resolution is entirely instrument limited) and often are not corrected for factors such as the wavelength dependence of the detection efficiency. The S1 SO fluorescence intensities for the three polycyclic aromatics summarized in Tables 1-3 show no dramatic differences in different environments, but subtle changes would not be apparent from the available data. The pentacene molecule, however, does seem to show some matrix-dependent vibronic intensity variations that may potentially be useful as a probe of environmentally induced skeletal distortions. The fluorescence spectra of pentacene in p terphenyl,62benzoic acid?l and PMMA2 exhibit three reasonably strong lines in the 1140-1190 cm-’ region whose relative intensities differ considerably among the three hosts. Based on approximate vibrational force field calculations, both we38 and others8* have concluded that one of the members of this triplet must be either an overtone or combination band or, more likely, a vibration that is nontotally symmetric in the D2h point group. A nontotally symmetric vibration may gain its intensity in the emission spectrum from crystal-induced distortions of the pentacene skeleton, which would presumably be different for different hosts and, possibly, for different sites within the same host. Such distortions have been discussed by others in relation to the differences in intersystem crossing rates among the four distinct spectroscopic sites (0’-04)of pentacene in p-terphen~l.~~-~~ C. Correlations between Electronic and Vibrational Inhomogeneous Distributions. While there has been recent theoretical interest in the question of correlations among
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inhomogeneous distributions for different spectroscopic transitions (e.g., to what extent do molecules having the same electronic transition frequency within an inhomogeneous en~-~~ semble also have similar vibrational f r e q u e n c i e ~ ? ) , ~there is a relative paucity of experimental data with which to compare, at least for aromatic molecules of the type thus far probed by single-molecule techniques. Several experiments have utilized fluorescence line-narrowing techniques to explore correlations between different electronic transitions (i.e., singlet-singlet and singlet-triplet, or two different singlet-singlet), leading to the general conclusion that the inhomogeneous distributions affecting different transitions are largely uncorrelated. For example, Griesser and Wild showed that narrow line width laser excitation into the SO S 2 transition of 1,3-dichloroazulene in a 3-methylpentane glass results in sharp, line-narrowed S2 SO fluorescence but broad S2 SI emission,21 indicating that molecules selected by their having a particular value of the S2SO frequency difference still possess a wide range of S2-S’ frequency gaps. Lee, Walsh, and Fayer showed that the “antiholes” that result from photophysical hole buming of pentacene in benzoic acid mixed crystals are very much broader than the “holes”, in this case comparing transitions involving the same molecule in two different environments related by proton ta~tomerism.~ They concluded that narrow band excitation of an inhomogeneously broadened electronic transition involves molecules having a very wide distribution of absolute energies and that the set of molecules having a single absolute energy in one state has a broad range of absolute energies in a second state. Correlations between electronic and excited-state vibrational transition frequencies have been explored through hole burning experiments that compare the widths of vibronic “satellite” holes with the resonant holes. In a variety of chromophores in glasses the satellite holes are consistently broader than the resonant holes even when irradiation into a vibronic transition is used to create satellite holes in the intrinsically narrower electronic origin, indicating incomplete correlation between electronic and excitedstate vibrational inhomogeneous distributions. 1 7 3 Information on correlations between electronic and ground-state vibrational frequency distributions, more germane to our work, should be available from fluorescence line narrowing, at least in glassy environments where the vibrational inhomogeneous widths are expected to be significant. However, we are aware of few such studies. Abram et al. mentioned that in line-narrowed spectra of perylene in an ethanol glass the mean ground-state vibrational frequencies appeared to vary by about 5 cm-l as the laser was tuned across the inhomogeneously broadened electronic origin band, l9 suggesting significant correlations between ground-state vibrational and electronic inhomogeneous distributions. Riebe and Wright showed that site selection on the electronic origin of pentacene in benzoic acid by fully resonant nondegenerate four-wave mixing gave narrowing of the vibronic transitions but not of the ground-state vibrational transitions, leading them to conclude that the vibrational and electronic inhomogeneous broadenings are not ~ o r r e l a t e d .Such ~ ~ questions are difficult to explore by fluorescence techniques in mixed crystals because the usual narrowness of the vibrational transitions requires extremely high-resolution detection of the spontaneous emission.
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3. Instrumentation and Experimental Methods for Vibrationally Resolved Single-Molecule Spectroscopy The general requirements for successful single-molecule detection were outlined in section 1B. Here we discuss more specifically the experimental configurations employed to date for the observation of vibrationally resolved single-molecule
10382 J. Phys. Chem., Vol. 98, No. 41, 1994
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spectra, as well as possible future modifications and improvements. The vast majority of single-molecule detection experiments have been performed using fluorescence excitation methods in which the electronic origin region is excited and total (Stokes shifted from the laser wavelength) fluorescence is detected as a monitor of absorption. Modification of the detection apparatus to spectrally disperse the fluorescence then gives the ground-state vibrational frequencies directly as the difference between the laser and emitted frequencies. We therefore discuss first the basic experimental requirements for the “parent” technique of spectrally undispersed single-molecule fluorescence excitation: low temperature, a tunable narrowband excitation source, a small illuminated sample volume, and highly efficient fluorescence detection with good stray light rejection. All high-resolution single-moleculespectroscopic experiments to date have been carried out at temperatures below 10 K. Typically this is achieved by holding the sample in a helium M C flow cryostat, which can routinely provide temperatures down Figure 3. Sample illumination and light collection geometry with in to about 1.5 K by pumping on the helium vapor. Nonetheless, situ focusing of the laser into the sample. S = sample, B = beam stop, it is clear that extremely narrow lines are not an absolute P = paraboloidal collection mirror, L = focusing lens, M = permanent requirement for success in single-molecule detection, as the magnet, C = coil (through which current is passed to make a variable recent near-field work at room temperature has dem~nstrated?~-~~ electromagnet). Selectivity for single chromophores can still be obtained with the laser spot size as small as possible. The experiments broader lines if the concentration of absorbers is reduced, and performed to date have used either single crystals or polymer the lower peak absorption cross section can be compensated to films having thicknesses of a few to perhaps a few hundred some extent by increasing the laser power, since broader micrometers. Two general approaches have been used to transitions also mean a higher saturation intensity. The achieve small spot sizes. The system described by Ambrose increased background emission with increasing laser intensity and Moerner uses a high-quality, short focal length lens, located is probably the limiting factor in determining how broad the inside the cryostat, to focus the laser spot onto the sample.30 excitation features can be and, correspondingly, how high a Variation of the current through a small electromagnet on which temperature can be tolerated in the absence of other processes the lens is mounted then allows optimal focusing (spot size as such as photochemical or photophysical hole burning. small as 5 pm in diameter) to be achieved in situ (see Figure In order to achieve optimal selectivity for individual mol3). An alternative approach is to attach the sample to the end ecules, and certainly if any detailed line shape studies are to be of a single-mode optical fiber having a core diameter of a few performed, the light source should be spectrally narrower than micrometers. This should result in spot sizes of less than 5 pn the homogeneous width of the electronic origin. However, the as long as a good contact is made between the sample and a powers required are low; with the small spot sizes generally cleanly cleaved fiber and the sample is not too thick. The laser used (see below), strong transitions can be nearly saturated with light is coupled into the optical fiber outside the cryostat. This only nanowatts to microwatts of power. Aromatic molecules method greatly simplifies the optical alignment since no focusing with relatively large oscillator strengths have lifetime-limited adjustments have to be made, while imposing the limitation that origin widths of tens of megahertz, line widths that are readily only a single physical region of the sample can be examined. achievable with commercial CW dye lasers. Slow laser drifts Collection of the fluorescence over a large solid angle is of the laser frequency can be a problem, and schemes based on generally achieved by using either a paraboloidal mirror (see either shifting the frequency axes of successive scans to correct Figure 3) or a high numerical aperture lens to approximately for drift31,45or actively locking the laser frequency to an external collimate the light and send it out of the cryostat. For detection referenceM have been utilized. Diode lasers have been utilized of total fluorescence, the collected light is focused into a in bulk hole burning experiment^^^,^^ and are an attractive option photomultiplier tube after passage through one or more longfor single-molecule experiments in the red and near-IR regions. pass filters to block scattered laser light, and photon counting No single-molecule experiments have yet been reported with electronics are used to process the signal. The spectral filtering excitation in the UV, where generation of tunable continuousused to reduce background always eliminates some of the real wave excitation is still rather difficult and is usually achieved fluorescence signal as well, particularly since those molecules by intracavity frequency doubling. Extracavity doubling of a that have the strong zero-phonon electronic origin absorption standing-wave single-frequency dye laser might, however, be line desirable for efficient excitation also have much of their a reasonable option in view of the very low powers required.g0 fluorescence in the origin transition, which is rejected along A very small illuminated sample volume is highly desirable with the scattered laser light. By the time the losses due to the when attempting to detect single molecules. Minimizing the finite solid angle of collection, reflection at the cryostat volume not only reduces the total number of illuminated windows, spectral filtering, the quantum efficiency of the photomultiplier, and other factors are taken into account, the chromophores (which could equally well be achieved by simply lowering the concentration of chromophores in the matrix) but system diagrammed in Figure 3 is estimated to end up detecting also minimizes the background emission due to impurity about 1% of the originally emitted photons.30 fluorescence and/or matrix Raman scattering. Furthermore, Detection of vibrationally resolved fluorescence requires that decreasing the spot size increases alA, the probability that the the emitted light, instead of being merely long-pass filtered, be single molecule selected will absorb a photon (see eq 2). dispersed with a spectrograph and detected as a function of wavelength. Even if all of the fluorescence could be refocused Therefore the samples should be made as thin as possible and
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I--LI
Figure 4. Sample illumination and light collection geometry with the sample attached to the optical fiber. S = sample, P = paraboloidal collection mirror, M = flat mirror (with small hole in center through which the fiber passes), B = 50% beam splitter, L = lens, LPF = long-pass filter, PMT = photomultiplier tube, CCD = charge-coupled
device detector. through the spectrograph entrance slit and the spectrograph had 100% throughput, the number of emitted photons per unit bandpass at the resolution desired for detailed vibrational analysis would be reduced from the “total” fluorescence signal by roughly 2 orders of magnitude even at the peak of the strongest emission lines. Therefore, if a reasonably large part of the fluorescence spectrum is of interest, efficient multichannel detection is essential. For experiments requiring detection in the mid-visible to red region of the spectrum, a cooled chargecoupled device (CCD) is presently the detector of choice. These devices have virtually no dark current, and currently available devices have quantum efficiencies in the 50-75% range around 500-650 nm. In order to maximize the detected signal the CCD should be mounted on a single spectrograph having a relatively high flnumber, although depending on the amount of scattered laser light and other light background it may be necessary to do some preliminary long-pass filtering of the collected light before coupling it into the spectrograph. The spectral resolution is determined both by the dispersion of the spectrograph and by the width of the entrance slit. Increasing the dispersion by using a longer focal length spectrograph and/or a higher groove density grating provides higher resolution (Le,, fewer wavenumbers per detector pixel), but it also reduces the total spectral coverage achievable with a detector of fixed physical array length. Figure 4 diagrams the sample illumination and detection setup used to obtain the single-molecule vibrational spectra in refs 43 and 46. The laser light (typically 5-10 nW at the sample from an argon-ion laser-pumped Coherent 599 standing-wave single-frequency dye laser having a line width of about 3 MHz) is brought into the cryostat through a single-mode polarizationpreserving optical fiber having a core diameter of about 4 p. The sample, in the form of a thin polymer film, is attached directly to the end of the fiber with a minimal amount of polycyanoacrylate adhesive. The tip of the fiber is positioned within the cryostat at the focus of a numerical aperture 1.0 paraboloidal reflector. The laser light transmitted through the sample passes through a small hole in the vertex of the paraboloid while the fluorescence is collected and collimated by the paraboloid, reflected 90” by a flat mirror, directed out of the cryostat, and divided by a 50% beam splitter. The transmitted light goes through a long-pass filter to a photomul-
tiplier with photon counting electronics as a detector of “total” fluorescence. The reflected light is focused with a 100 mm focal length lens onto the entrance slit of a 0.75-m single spectrograph equipped with a 1200 groove/mm ruled grating and detected with a Princeton Instruments thinned, backilluminated, liquid nitrogen-cooled CCD. In some of the experiments reported in section 5, optical alignment of the emission was facilitated by adding a small amount of perylene to the polycyanoacrylate adhesive used to attach the sample to the optical fiber. Irradiation with a He-Cd laser at 441 nm then caused the perylene to emit brightly in the green, providing a strong alignment beam that could be followed by eye all the way to the entrance slit of the spectrograph. The high dispersion of the detection system provides about 6 cm-’ spectral resolution at detection wavelengths near 600 nm even with a rather wide 200 pn entrance slit, but only about 400 cm-’ of the spectrum can be viewed at one time. The stray light rejection is sufficiently good that no additional filters are usually required to block scattered laser light, even when detecting within 150 cm-’ of the laser frequency. The procedure used to obtain single-molecule dispersed fluorescence spectra is as follows. First, the dye laser frequency is scanned over regions of typically 20-50 times the singlemolecule line width (usually about 10 GHz for terrylene in polyethylene) while monitoring total fluorescence until a strong, stable, well-isolated single-molecule feature is identified in the excitation spectrum. The laser frequency is then tuned to the peak of the excitation feature, the spectrograph is set to the desired detection wavelength, and a signal is collected on the CCD for one or more accumulations of typically 120 s each, while the total fluorescence signal is simultaneously monitored to ensure that the molecule has not yet undergone any hole burning or spectral diffusion process that takes it out of resonance with the laser. Such “irreversible” absorption frequency changes are typically what limit the length of time over which dispersed emission can be accumulated and thus the quality of the spectra. In the system in which our first experiments were performed, pentacene in p - t e r p h e n ~ l most ,~~ of the molecules have high photophysical stability but the laser frequency can drift significantly relative to the very narrow single-molecule line width during the course of data accumulation. These frequency drifts on the tens of megahertz scale were compensated manually during data collection by tuning the laser to keep the total fluorescence signal maximized. For terrylene in polyethylene, spectral diffusion of the molecular transition frequency was much more significant than the instability in the laser frequency. Although efforts to manually tune the laser to “chase” a single molecule’s frequency jumps were usually not very successful, spectra of reasonable quality could be obtained despite the limited time the molecule remained on resonance with the laser. Note that the frequency range over which the laser might drift during a measurement, or over which it might be deliberately tuned in attempting to follow a spectrally diffusing molecule, is considerably less than 1 GHz (0.03 cm-’), which is completely negligible relative to the resolution of the emission spectrum (typically about 6 cm-’). The laser frequency is measured with a wavemeter, while the dispersed emission spectra are calibrated in frequency using a variety of dye laser lines.
4. Single-Molecule Vibrational Spectroscopy of Pentacenelp-Terphenyl The first single-molecule vibrational spectra38were reported on the “classic” mixed crystal system of pentacene inp-terphenyl discussed briefly in section 1C. These preliminary experiments
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10384 J. Phys. Chem., Vol. 98,No. 41, 1994 r
t
TABLE 4: Calculated and Observed Electronic Origin Shifts for MonoJ3C Substituted Pentacend calcd
bosition, 0.17 (Ci, 4) 0.19 (cis, 4) 0.21 (CIS, 4) 800
1200
1600
2000
Wavenumber (cml)
Figure 5. Vibrationally resolved fluorescence spectra of pentacene in p-terphenyl. (a) “Bulk”spectrum (excitation near peak of 0 1 electronic origin). (b) Spectrum of one single molecule. (c) Signal obtained with the laser tuned out of resonance with the molecule (b). (d) Spectrum of another single molecule. 150
30
I
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‘
0
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I
1
8
1
I
1
I
100
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300
400
500
Relative frequency (MHz)
Figure 6. Total fluorescence excitation spectra of pentacene in p-terphenyl. The two overlapping curves in each case are traces obtained before and after measurement of the dispersed fluorescence spectra of Figure 5. Labels b-d indicate the laser frequencies at which the corresponding single-molecule fluorescence spectra of Figure 5 were obtained. utilized the sample mounting, illumination, and light collection apparatus depicted in Figure 3. The fluorescence was then beam split and sent to the long-pass filter/photomultiplier and spectrograph/CCD detectors. A relatively low-dispersion spectrograph was used for these experiments, allowing a large portion of the emission spectrum to be viewed at once but also limiting the resolution of the vibrational spectrum and requiring that a narrow entrance slit be used. Figure 5 compares the lowresolution “bulk” dispersed fluorescence spectrum of pentacene in p-terphenyl at 1.8 K with the spectra of two different single molecules whose total fluorescence excitation spectra are shown in Figure 6. While the signal-to-noise ratio of the singlemolecule spectra is low, it is clear that they both arise from pentacene. In addition, when the laser is detuned from resonance with a fluorescence excitation feature by about 50 MHz in Figure 5c, the vibrational spectral features nearly vanish, demonstrating that the spectra of Figure 5 parts b and d do arise almost entirely from single molecules. These spectra, while quite preliminary, demonstrated the feasibility of the technique and suggested several directions that could be interesting to pursue. One is the possibility of using the frequencies or, more likely, the vibronic intensities to probe slight differences in the local environments of different molecules in the crystal. Since the inhomogeneously broadened vibrational line widths in this system are much less than 1 cm-l, observation of frequency differences between molecules would require extremely high resolution which may be incompatible with the highly efficient detection needed for single-molecule studies. Intensity variations among molecules are easier to detect at moderate resolution and may be both larger and more informative than frequency differences. As mentioned in section 2B, the relative intensities of the triplet of lines in the 1140-
obsd
(re1 intensity)’ 0.11 (2.8) 0.30 }(8.1) 0.33
:::;
obsdd 0.11,0.12 0.29,0.30 0.33.0.34 0.49, 0.50 0.51,0.52
}(3.2) 0.35 0.33 (Ci4.4) (C2,4) 0.65 (2.0) 0.65,0.66 0.77 (c13, 2) All shifts (in cm-’) are to higher energies relative to all-I2C pentacene. Calculated using QCFF/PI+CISD method. Carbon atoms are labeled as in ref 117. Degeneracy is number of equivalent carbon atoms; relative intensities should be proportional to degeneracies for random 13C-substitution. Reference 92. Reference 94; frequency shift data for both 0 1 and 0 2 origins.
1190 cm-l region (not well resolved in Figure 5 ) appear to be sensitive to the nature of the environment and are possibly related to out-of-plane distortions of the pentacene skeleton. Vibrational frequency and intensity differences among molecules could also arise from isotopic substitution. Given the natural abundance of I3C, the purely statistical probability that a pentacene molecule will contain at least one 13Catom is 22%. Most of the strongly Franck-Condon active skeletal vibrations of pentacene are fairly delocalized, and the shifts due to a single I3C substitution are generally expected to be only a few wavenumbers, but shifts of this magnitude should be detectable with modest improvements in the data over those obtained in this preliminary work. Isotopic substitution also breaks the molecular symmetry and is calculated to produce some readily detectable intensity changes.38 Any heavy atom substitution should, in general, cause a small blue shift of the electronic origin, and since our experiments utilized excitation on the red side of the lowest-energy (01)site, our chances of encountering a 13C-substitutedmolecule were greatly reduced over the purely statistical 22%. Calculations using the QCFFP1-I-CISDmethodg1 predict a blue shift of the origin ranging from 0.17 to 0.77 cm-’ for the six possible distinct I3C substitutions. The crystals used in much of the single-molecule work on pentacene have electronic inhomogeneous line widths that considerably exceed these shifts, so the inhomogeneous distributions of the various isotopic species will be significantly overlapped. On the other hand, crystals that are grown and handled very carefully to avoid strain can have inhomogeneous electronic origin widths smaller than the isotopic shifts, and indeed several g r o ~ p s ~ have ~-~~ recently observed in bulk fluorescence excitation spectral features that may be due to resolved electronic origins of natural abundance 13C substituted pentacenes. The observed and calculated intensities and shifts are summarized in Table 4. The calculated shifts are fairly sensitive to the level of theory used and should not be considered very accurate but do show a reasonable qualitative agreement with experiment. These results suggest the amusing possibility that vibrational spectra of isotopically substituted large molecules, which can be very useful in the refinement of empirical force fields, can be obtained from high-resolution spectroscopy without the need to synthesize specifically labeled compounds. 5. Single-Molecule Vibrational Spectroscopy of Terrylend Polyethylene A. Experimental Single-Molecule versus Bulk Spectra. The first, and thus far the only, detailed single-molecule vibrational spectroscopy has been carried out on the tenylene in polyethylene system introduced in section 1D. The experimental apparatus used for these studies was shown in Figure 4.
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200
300 400 Wavenumber (cm-’)
500
Figure 7. Vibrationally resolved fluorescence spectra of terrylene in polyethylene. The “bulk” spectrum was obtained with excitation near the peak of the inhomogeneouslybroadened origin band. A-E represent different single molecules.
Thin f i h s containing IO+ or mass fraction terrylene were prepared by adding a solution of terrylene in methylene chloride to polyethylene powder, removing the solvent in a vacuum oven, melting a small amount of the resulting powder, pressing it between a quartz flat and a microscope slide, and quenching rapidly in liquid nitrogen. The goal was to obtain samples that were as amorphous as possible in order to minimize scattered laser light, but no quantitative measure was made of the degree of crystallinity of the final films. The resulting samples showed an inhomogeneously broadened fluorescence excitation spectrum with a maximum at 569 nm and a full width at half-maximum of about 150 cm-’. Single-molecule vibrational spectra were obtained with excitation wavelengths both to the red and slightly to the blue of the A,,,=. Cleanly isolating single molecules at bluer excitation wavelengths is complicated, however, by interferences from the phonon sidebands of redder-absorbing molecules. For this reason a “nonresonant” spectrum was obtained after each single-molecule spectrum, either by waiting until the single-molecule fluorescence excitation feature had vanished due to spectral diffusion or hole burning or by tuning the laser several gigahertz away from resonance. In cases where the nonresonant spectrum showed any discemible vibrational features, this spectrum (which was at most 20% as intense as the resonant spectrum) was subtracted from the single-molecule signal. Figures 7 and 8 compare the single-molecule and “bulk” emission spectra in two different frequency regions. The majority of the approximately 50 single molecules examined (e.g., A-C in Figure 7 and A, F, and G in Figure 8) exhibit vibrational frequencies and intensities very similar to those of the bulk sample except for the considerably stronger phonon sidebands in the bulk spectrum. These are attributed to significant excitation of phonon sum bands in the bulk spectrum due to saturation of the zero-phonon resonances andor preferential hole burning of the origin-resonant molecules at the higher laser intensities employed for the bulk spectrum. The singlemolecule spectra were obtained at much lower intensities and
1300 1400 1500 Wavenumber (cm-’)
1200
1600
Figure 8. Same as Figure 7 in the “fingerprint”region of the spectrum.
200
300 400 Wavenumber (cm-’)
500
Figure 9. Comparison of the emission spectra obtained with the laser set to the peak of a single-molecule resonance in the total fluorescence excitation spectrum (same as A in Figure 7) and with the laser tuned about 1 GHz away from resonance. The two traces are offset vertically but have the same vertical scale and represent equal accumulation times. arise from excitation of origin bands only. Detuning of the laser from resonance with molecule “A’ by 1 GHz yields a featureless spectrum (Figure 9), demonstrating that, at least in the red region of the excitation spectrum, all of the observed vibrational features arise from the resonant single molecule. The bulktype spectra, which we denote “type l”, are characterized by a very strong low-frequency line at 243 cm-’, a line near 488 cm-’ which we interpret as its overtone, a strong doublet near 1272 and 1283 cm-’, a weaker doublet near 1514 and 1529 cm-’ which we assign as the combination band of the 12721 1283 doublet with the 243 cm-’ transition, and a strong line near 1562 cm-’. Possible assignments of these lines to specific vibrations are discussed in the following section. In addition to these “normal” type 1 molecules, a few molecules were observed with a qualitatively different frequency and intensity pattem. These “type 2” molecules (D and E in Figure 7 and E and H in Figure 8) have their most intense line shifted down in frequency by nearly 30 cm-’ relative to the type 1 molecules, with the overtone and combination bands involving this transition also shifted accordingly. The strong line near 1562 cm-’ in the type 1 spectra is found at a slightly lower frequency, and there is only a single line near 1272 cm-’ rather than a doublet. The bulk spectrum, while it clearly resembles most closely the type 1 spectra, does exhibit a very weak line at the type 2 frequency near 216 cm-’ (barely discernible in Figure 7). While we did not examine enough individual molecules to obtain very
10386 J. Phys. Chem., Vol. 98, No. 41, 1994
Myers et al.
TABLE 5: Calculated and Experimental Vibrational Frequencies of TerrylenP calcd freq (cm-’) exptl freq (cm-l)b calcd displacement‘ description 1630 0.27 stretch of CC bonds parallel to long axis; end units in phase relative to center naphthalene 1562 (s) 1622 stretch of CC bonds parallel to long axis; 0.33 end units out of phase relative to center naphthalene 1593 CC stretch of bonds adjacent to outer CC bonds parallel 0.09 to long axis in the end units and of the central CC bond in middle unit 1499 0.03 CC stretch in all units, inner one out of phase relative to outer ones 1451 1370 (w) 0.02 CC stretch of outer CC bond adjacent to middle CC bond in end units and of CC outer bonds parallel to long axis 1385 1358 (m) 0.04 CC stretch mostly of inner CC bond of all units 1365 1312 (m) 0.12 CC stretch 0.25 CC stretch of bond parallel to long axis, mainly on middle unit 1333 1283 (s) 0.74 1313 1272 (s) CC stretch of bond parallel to long axis, mainly on end units 1213 0.12 CC stretching and CH rocking CH rocking in all units 1165 0.01 1110 0.04 CC stretch in outer units, CH rock in middle 0.05 CC stretch and CH rock 1089 1037 (m) 0.01 880 830 (w) ring breathing, inner unit out of phase relative to two outer ones 834 780 (w) 0.05 ring deformation of outer naphthalenes 606 584 (m) 0.15 short axis stretch of middle naphthalene, CCC bend along middle CC bond of ends 554 536 (s) 0.23 short axis stretch of end naphthalene 0.10 CCC bend along middle bond of end units and short axis stretch 484 439 (w) of middle unit 248 243 (vs) 0.53 long axis stretch of whole molecule Calculated frequencies and intensities from QCFF/PI+CISD method. All 19 total symmetric in-plane vibrations excluding CH stretches are listed. Experimental frequencies in “bulk” spectra. Qualitative intensities are listed in parentheses. Absolute magnitude of difference between ground and excited state equilibrium geometry in dimensionless normal coordinates of the ground state. good statistics, our single-molecule spectra suggest that about 10-20% of the molecules in the sample are type 2, while the type 2 contribution to the bulk spectrum is clearly much smaller than this. We believe that this results from a generally lower photophysical stability of type 2 molecules coupled with the use of higher laser intensities to record the bulk spectra. Reduced photophysical stability for the type 2 molecules was not quantitatively documented but was suggested by the qualitative observation that it seemed unusually difficult to find type 2 molecules that would remain on resonance with the laser for long enough to record a high quality vibrational spectrum. The type 2 molecules therefore make a smaller contribution to the bulk spectrum than expected based on their statistical distribution because they undergo more rapid spectral hole burning processes that remove them from resonance with the laser. We have attributed both the type 1 and type 2 spectra to terrylene molecules. The possibility that the type 2 spectra arise from a chemically different species cannot be entirely discounted but appears quite unlikely. The chemical impurity would have to possess a very strong, narrow electronic absorption in the same spectral region as terrylene, a high fluorescence yield together with very low yields for triplet formation and photochemical or photophysical hole burning, and a vibrational spectrum very similar to terrylene’s. About the only reasonable possibility might be a methyl- or other alkyl-substituted terrylene, and even such a species is likely to have its electronic origin shifted from terrylene’s by a significant fraction of the inhomogeneous line width, whereas most of the type 2 molecules observed had their electronic origins fairly close to the inhomogeneous band center. Normal mode calculations were not consistent with alkyl substitution as a cause for the vibrational frequency changes observed in the type 2 spectra. Finally, it is highly unlikely that our samples contain as much as 10-20% of an impurity. The most consistent explanation is that the type 1 and type 2 spectra represent terrylene molecules
in two very different types of environments within the polyethylene matrix. B. Comparison of Experimental and Calculated Spectra. Analysis of the single-molecule spectra is considerably hindered by the fact that terrylene is an almost unknown molecule spectroscopically. The bulk and single-molecule fluorescence spectra we have obtained in polyethylene are, to our knowledge, the only vibrational spectra of any kind yet reported for terrylene. Therefore, our efforts to assign and interpret our single-molecule spectra have been based entirely on comparison with calculations. Planar terrylene belongs to the D2h point group and has 23 totally symmetric (a,) vibrations. We anticipate that only the totally symmetric modes will be strongly Franck-Condon active in the origin-excited emission from this strongly allowed transition, although distortions away from D2h symmetry due to interactions with the matrix might induce intensity in nominally nontotally symmetric modes. Initial vibrational calculation^^^ were carried out by using the semiempirical force field developed by Ohno to fit in-plane vibrations of a series of polycyclic aromatic hydrocarbon^.^^ These are of some help in assigning the spectra, but they are strictly ground-state calculations and provide no information about which of the totally symmetric modes are expected to be most intense in our fluorescence spectra. More recently we have carried out more informative calculations by utilizing the QCFFPI+CISD meth0d9l to generate equilibrium geometries and normal modes for both the ground and the first excited state, from which vibrationally resolved absorption and fluorescence spectra can be calculated. The frequency and intensity information together provide a better, although still preliminary, basis on which to assign our spectra. The lowest singlet electronic transition in terrylene is calculated to be essentially a HOMO to LLJh40 excitation polarized along the long axis of the terrylene molecule. Table 5 shows the calculated vibrational frequencies, normal mode descriptions, S1 SO displacement parameters, and most
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I . 3 . l 600 1000 1400 Wavenumber (“1)
.
I 1800
Figure 10. SIorigin SOfluorescence spectrum of terrylene calculated using the QCFFPI-CISDmethod. A line width of 5 cm-I has been used.
probable corresponding experimental frequency for the 19 nonCH-stretching ag vibrations. The fluorescenceintensity resulting from excitation into the electronic origin is approximately proportional to the square of the displacement parameter (exactly proportional if the ground and excited state normal modes have identical frequencies and normal mode descriptions). While the calculated frequencies are uniformly too high, there is a fairly good correspondencebetween calculated and observed frequencies and intensities, particularly in the low-frequency region. Most importantly, the assignment of the 243 cm-’ vibration in the bulk spectrum to an overall long-axis stretch seems unambiguous (the Ohno force field calculation similarly predicts an overall long-axis stretching mode at 225 cm-’ as the only totally symmetric fundamental below 400 cm-’). The assignments of the 488 cm-’ line as the overtone of this vibratioll with a small positive anharmonicity and of the 1514/1529 cm-’ doublet as its combination band with the 1272/1283 doublet also seem clear. The observed line at 1562 cm-’ almost certainly corresponds to one or both of the highest-frequency CC stretches calculated at 1622 and 1630 cm-’. The assignments are less definite in the mixed CC stretching/CCH rocking region from 1250-1400 cm-’. The rather broad band at 1300 cm-’ in the bulk spectrum may be largely or entirely a phonon sideband, but the other five bands at 1272, 1283, 1312, 1358, and 1370 cm-’ are all observed in at least some of the singlemolecule spectra and have no obvious assignments as overtones or combination bands. All of these vibrations can reasonably be assigned as totally symmetric fundamentals, although there remains the possibility that nontotally symmetric fundamentals could appear with intensity due to distortions of the terrylene skeleton away from D2h symmetry. The “unusual”, type 2 single-molecule spectra are actually in better accord with the calculations. The strong line near 215 cm-’ is presumably the mode calculated at 248 cm-’, with its overtone observed near 428 cm-l. The similar intensity ratios of 428:212 cm-’ in molecule E and 490:243 cm-’ in molecules A-C (Figure 7) further support the assignments of these two bands as the overtone and fundamental of essentially the same vibration in both cases. The combination band of the lowfrequency mode with the 1272 cm-’ mode appears near 1490 cm-I in type 2 spectra. Type 2 molecules exhibit only two strong lines in the 1250-1400 cm-l region, quite consistent with the calculation. The calculated fluorescence spectrum of Figure 10 shows that while the agreement is far from quantitative, the overall intensity patterns appear more similar to those of the type 2 than the type 1 molecules. C. “Type 1” and “Type 2” Spectra: Tentative Assignment to Crystalline and Amorphous Environments. The data shown in Figures 7 and 8 clearly indicate that our rapidly quenched samples of terrylene in polyethylene contain two distinctly different types of terrylene molecules. It is also well
established that polyethylene solidifies as a polycrystalline matrix in which small crystalline domains are connected by amorphous regions.96 This suggests that the type 1 and type 2 spectra correspond to terrylene molecules in these two different environments. We have tentatively assigned the type 1 molecules to those in or on the surface of the crystalline regions and type 2 molecules to those in the amorphous regions.43 This assignment is based on a combination of vibrational spectroscopic and photophysical observations. As discussed above, the type 1 molecules show more vibrations in the “fingerprint” region of the spectrum than expected, suggesting that they may be conformationally perturbed; we expect that the crystalline domains, being more dense and more ordered, are more likely to conformationally distort a guest molecule. The much higher frequency of the strong low-frequency mode in type 1 compared with type 2 molecules is also consistent with location of the type 1 molecules in a more dense environment that provides less free volume. This vibration, involving overall long-axis breathing, should have a larger “activation volume” than most or all of terrylene’s other vibrations. Finally, the apparently lower photophysical stability of the type 2 molecules (more spectral diffusion and hole burning) also seems qualitatively consistent with their residence in a more amorphous environment. The very recent results on terrylene in a hexadecane Shpol’skii matrixS2 qualitatively support the expectation that terrylene shows higher photophysical stability in crystalline than in amorphous environments of very similar chemical composition. This assignment must still be considered quite tentative and requires further corroborating evidence. Varying the degree of crystallinity of the polyethylene matrix by changing either the quenching conditions or the molecular weight or degree of branching of the polyethylene is one possibility, but the results would not be conclusive because we are able to obtain vibrational spectra for only the most photostable terrylene molecules, which are already a self-selected subset of the ensemble probably weighted heavily toward the more photostable, type 1, molecules. A better alternative might be to obtain spectra in environments that are chemically similar to polyethylene but better defined as either crystalline or amorphous. The terrylene in hexadecane Shpol’skii system should be a very good model for the crystalline regions of polyethylene. Models for the amorphous region may be found among the purely amorphous aliphatic hydrocarbon polymers such as atactic poly(propylene), and the probably lower photophysical stability in such environments would not be a problem because there is no need to observe spectra of single molecules in order to establish the frequency and intensity patterns characteristic of an amorphous environment. Raman spectra of terrylene in liquid solution could also provide the frequencies of the totally symmetric vibrations in an amorphous environment, but such measurements are problematic due to the very poor solubility of terrylene and its strong fluorescence. D. Electronic-Vibrational Frequency and Intensity Correlations. As discussed in section 2, the degree to which the frequencies of different inhomogeneouslybroadened transitions in a condensed phase are correlated has been a topic of experimental and theoretical interest for some time. Since single molecules are selected by their electronic origin frequency within the inhomogeneous ensemble, single-molecule vibrational spectra allow correlations between electronic and ground-state vibrational frequencies, between electronic frequency and vibronic intensities, and among the frequencies of different vibrations to be probed in unprecedented detail. We first address the correlation between electronic origin
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10388 J. Phys. Chem., Vol. 98, No, 41, 1994 i
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Figure 13. Correlation between electronic origin wavelength and intensity ratio of the 1283 to 1271 cm-' lines of type 1 terrylene molecules in polyethylene.
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Figure 11. Correlation between electronic origin wavelength and vibrational frequencies of type 1 terrylene molecules in polyethylene.
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Figure 12. Fluorescence excitation spectrum of terrylene in polyethylene. Points represent the intensity, normalized to the incident laser power, of the 243 cm-' line in the fluorescence spectrum as a function of excitation wavelength. The laser frequency was not fixed but was allowed to scan freely over a range of approximately 15 GHz about each nominal wavelength, thus averaging the fluorescence over aIl molecules having resonances within this bandwidth. The bars along the x-axis indicate the excitation wavelengths at which single-molecule fluorescence spectra were obtained. frequency and ground-state vibrational frequencies. We limit our examination to type 1 molecules (see above) because the number of type 2 spectra measured was too small to be statistically significant. Figure 11 is a plot of the frequencies of the three most intense ground-state vibrations versus electronic origin wavelength. Clearly there is a slight tendency for the molecules absorbing far to the red (beyond 575 nm) to have higher frequencies for all three of these vibrations. The molecules absorbing between 575 and 578 nm have average vibrational frequencies of 245.2 f 3.3, 1273.0 f 1.6, and 1563.0 f 1.7 cm-' for these three strongest vibrations, while the molecules absorbing between 566 and 575 nm have average frequencies of 242.6 3~ 1.3, 1269.6 & 1.6, and 1559.3 f 1.2 cm-I. Examination of the excitation spectrum in Figure 12 shows that very few molecules absorb to the red of 575 nm, so these represent molecules in highly unusual environments. We suggest that these far-red-absorbing molecules may be those residing in unusually tight "cages" in the matrix.& For strongly allowed electronic transitions of nonpolar molecules in nonpolar environments, the vapor-to-condensed phase electronic frequency shift is usually dominated by the dispersion interaction (induced dipole-induced dipole), which gives an increasing red
shift of the electronic spectrum with increasing polarizability (e.g., density) of the medium. Other types of experiments (e.g., hole burning/Stark effect) on related chromophores in polymeric glasses also suggest tighter solvent cages for redder-absorbing molecules.10 Vibrational frequencies, on the other hand, may shift either up or down in frequency with increasing density depending on whether the attractive (dispersion) or repulsive part of the solvent-solute interaction dominates;97presumably the repulsive forces are most important in this case. While the vibrational frequencies reflect only properties of the electronic ground state, the vibronic intensities also depend on the interactions of the electronically excited chromophore with its environment. The line near 1283 cm-' has essentially no intensity in type 2 molecules, and its intensity also varies considerably among different type 1 molecules. This behavior is mirrored in the results of the QCFFPI-tCISD calculations (Table 5), in which the displacement parameters for the two totally symmetric fundamentals calculated near 1333 and 1313 cm-l are unique in depending strongly on the details of the calculation (e.g., whether single or single plus double configuration interaction is used), suggesting that they may also be sensitive to small distortions of the terrylene skeleton or variations in the nature of the environment. Figure 13 plots the 1283:1271 cm-' intensity ratio for type 1 molecules as a function of origin wavelength. There is perhaps a slight degree of correlation, with the lower intensity ratios tending to be found in the redder-absorbing molecules, but far more data are needed to establish this. Although exploration of electronic-vibrational frequency and intensity correlations does not require single-molecule techniques, the ability to interrogate single molecules greatly increases the level of detail with which such correlations can be probed. Molecules selected by their electronic origin frequencies can also be examined for correlations between properties that do not directly involve the origin frequency, such as vibrational spectra and radiationless relaxation rates. Singlemolecule spectroscopy is a promising tool because sites can be characterized by more than one nonaveraged parameter and multidimensional correlations among them can be studied.
6. Conclusions and Prospects Since we have stressed the ground-state vibrational information contained in spectra such as those displayed in Figures 7 and 8, it is natural to question whether they should be considered as resonance Raman, fluorescence, or a mixture. It might seem that one could easily distinguish the two by a simple gated detection method in which the detection window is delayed from an excitation pulse to reject prompt Raman scattering, but the situation is not so simple. While there is no uniformly accepted terminology, "fluorescence" generally refers to a process in
Feature Article which the absorption and emission steps can be considered as distinct with emission occurring from an incoherent population in the electronically excited state, while “resonance Raman scattering” is a two-photon process in which the absorption and emission events cannot be ~ e p a r a t e d . ~ ~Since - ’ ~ both fluorescence and Raman are “incoherent” spectroscopies in the sense that contributions from different molecules simply add at the signal level and do not interfere, these distinctions apply equally well to experiments on single molecules. For continuous, monochromatic excitation, true fluorescence can occur only if there is some source of electronic pure dephasing, competitive with the excited-state radiative decay, which converts the lightdriven electronic coherence into a population. Therefore the emission from pentacene in p-terphenyl, which exhibits a lifetime-limited origin line ~ i d t h , should ~ ~ , ~properly ~ be considered as resonance Raman scattering if measured with a laser whose line width is much narrower than the 8 MHz width of a single molecule’s origin. The situation is less clear for perylene or terrylene in polyethylene, because while the observed single-molecule origin line widths significantly exceed the presumed lifetime limit, these apparent widths may arise almost entirely from spectral diffusion on time scales much longer than the lifetime. The distinction between the two processes is largely moot, however, since the spectroscopically interesting differences between resonance Raman and fluorescence arise from interferences between different excited-state levels in the Raman process. When the electronic spectrum is so sparse that only a single vibronic level is near resonance at a given excitation frequency, such interferences are insignificant even if the emission is, in principle, of the Raman type. Direct measurements of emission lifetimes necessarily use pulsed (or modulated) excitation sources. This fundamentally changes the physics of the process by imposing on the excitation source a spectral line width which, in order for useful lifetime measurements to be made, must be on the order of or greater than the natural line width of the molecular excitation feature. This loss of spectral purity produces what has been termed “light-induced dephasing”,lO’causing the molecular emission to have the characteristics of true fluorescence even in the absence of any material pure dephasing. Thus, the fact that time-resolved emission from single molecules of pentacene in p-terphenyl acts like “normal” fluorescence (nearly single exponential decay with the expected lifetime)39is not relevant to characterization of the narrow-band excited emission as Raman or fluorescence. There may, however, be some interesting physics to be learned by combining time- and frequencyresolved emission measurements, as discussed more completely with respect to ensembles of molecules in refs 102 and 103. Such experiments could be carried out by using very dilute samples (to allow selective single-molecule excitation with a necessarily broader-bandwidth pulsed laser) and combining pulsed laser excitation with a gated or gain-modulated intensifier before a CCD detector. A similar system has been demonstrated for fluorescence lifetime imaging of cells, where the CCD is used for capturing a spatial image rather than for wavelength multiplexing.104 The field of single-molecule spectroscopy remains, at present, mostly a set of proof-of-principle experiments and demonstrations of feasibility. However, the experimental results on spectral diffusion, in particular, are starting to feed back in a rewarding way into theoretical developments on the structure and dynamics of both crystalline and glassy solids.86J05-’07 Approaches that combine more than one type of spectroscopic observation at the single-molecule level-e.g., vibrational spectroscopy together with Stark effects or spectral diffusion
J. Phys. Chem., Vol. 98, No. 41, 1994 10389 measurements-have great potential for truly local probing of chromophore-environment interactions in condensed matter. The main obstacle thus far has been the relative complexity of the probe molecules (e.g., terrylene) employed. Clearly the ideal situation would be to apply single-molecule techniques to chromophores whose spectroscopy is better understood. Most of the potential smaller molecule candidates absorb only in the UV where the experiments are technically much more difficult, but they should be possible, and the relatively large signals obtained from the chromophores thus far studied suggest that these experiments can also succeed with chromophores having less ideal photophysical parameters. Single-molecule techniques are going to be most valuable for application to systems in which inhomogeneous broadening is unavoidably severe. Biopolymers, especially chromophoric proteins, are one intriguing example. While the “structure” of most functional proteins is relatively well-defined in a crude way, proteins in solution and even in crystals have a very large number of usually similar conformations that are close in energy but separated by significant energetic barriers, and the consequent distribution of conformational states shows up in both the spectroscopy and the functional kinetics.108-112The possibility of making spectroscopic or, better yet, functional measurements on one molecule at a time is tremendously attractive. Another area where structural inhomogeneity is a major problem is in molecular clusters such as the dye aggregates113,’l4 and colloidal semiconductor particles’ 1 5 ~l6 1 whose optical properties have attracted great recent interest. The inevitable distribution of aggregate sizes and geometries in such systems often seriously compromises efforts to understand and model their photophysics. The possibility of making a number of spectroscopic measurements on one particular aggregate at a time is very appealing. Few chromophoric proteins or molecular aggregates possess the properties of strongly allowed and narrow homogeneous origin transitions, no significant photophysical bottlenecks, strong fluorescence, and very low photophysical and photochemical hole burning yields that have thus far been necessary for high-resolution single-molecule spectroscopy to succeed. However, representatives of these interesting classes that do meet these criteria may exist, and it is equally likely that slightly different approaches will allow probing of single molecules or individual aggregates which do not meet all these criteria.
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