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J. Phys. Chem. B 1997, 101, 8054-8058
ARTICLES Size-Dependent Fluorescence Emission Spectra and Lifetimes of Microcrystals of the Dye N,N′-Bis(2,5-di-tert-butylphenyl)-3,4:9,10-perylenebis(dicarboximide) (DBPI) Studied by Confocal Fluorescence Microscopy Prem B. Bisht,† Kazuhiro Fukuda, and Satoshi Hirayama* Laboratory of Chemistry, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan ReceiVed: March 20, 1997; In Final Form: June 4, 1997X
The time-correlated single-photon counting (TCSPC) technique combined with confocal fluorescence microscopy has revealed the nonexponential character of the fluorescence decay (0.1-10 ns) for highly fluorescent single microcrystals of the dye N,N′-bis(2,5-di-tert-butylphenyl)-3,4:9,10-perylenebis(dicarboximide) (DBPI) of a size as small as a few micrometers. Steady-state and time-resolved emission spectra of the microcrystals have also been measured with a fluorescence microscope. A size dependence is observed for both fluorescence emission spectra and lifetimes. The fluorescence decay profiles of the microcrystals, when observed with confocal optics, exhibit a dominant contribution of a short-lived (0.11-0.14 ns) component. From these results it has been concluded that the self-absorption effect is playing a major role in characterizing the fluorescence of microcrystals of DBPI with no contribution from any new emitting species.
1. Introduction Fluorescence microscopy is widely used in the biological, physical, and chemical disciplines due to its resolution and sensitivity. In particular, the high spatial resolution attained by confocal scanning fluorescence microscopy has revolutionized the field of multidimensional image reconstruction.1a Nearfield scanning optical microscopy1b (NSOM) recently has been employed to spatially resolve mesoscopic inhomogeneous spectral features in small crystals2 and J-aggregates3,4 of dyes by Barbara’s group. NSOM can be used to characterize samples on the tens of nanometers distance scale.5 Actually, singlemolecule detection limits have been reached by NSOM.6 On the other hand, if the sample of interest (usually highly spherical or cylindrical in shape) possesses morphology dependent resonances (MDR), the size should be limited to well above 1 µm to a few tens of micrometers due to the dependence of the MDR on radius and the refractive index of the microparticle.7,8 Therefore, depending upon the phenomena and samples of interest, one has to chose a spatial resolution that is convenient for measurements with the required signal-to-noise ratio. Microcrystals of fluorescent dyes are subjects of recent study in the field of fluorescence microscopy since fluorescence properties are strongly dependent on their size, shape, structures, manners of packing, defects, and other irregularities.2 Perfectly cylindrical shaped dye microcrystals of a few micrometers diameter can also exhibit MDR due to their presumably higher refractive index and, thus, can influence the radiative rate. The dye N,N′-bis(2,5-di-tert-butylphenyl)-3,4:9,10-perylenebis(dicarboximide) (DBPI) has a fluorescence quantum yield of unity, has high photostability in solution, and shows laser activity in condensed phase.9-11 While working with the microdroplets * To whom all correspondence should be addressed. † Permanent address: Department of Physics, Indian Institute of Technology Madras 600 036, India. X Abstract published in AdVance ACS Abstracts, September 15, 1997.
S1089-5647(97)01008-0 CCC: $14.00
of DBPI during an earlier work,7,8 we found that under continuous irradiation of the laser, due to the longer data acquisition time required for the droplets, the microdroplets get heated causing evaporation of solvent molecules. This resulted in the formation of the microcrystals of the fluorophore in the droplet, which caused severe problems in fluorescence decay measurements. A series of the fluorescence lifetimes could be obtained depending upon the size and shape of the microcrystals. We found it compulsory, therefore, to thoroughly study the size effect on the fluorescence emission spectra and lifetimes of the microcrystals of DBPI. By introducing confocal optics into a conventional fluorescence microscope, it is possible to improve spatial resolution up to ∼0.5 µm. The resolution of space of half a micron can be sufficient to study phenomena such as “active shell” effects of MDR on the radiative emission rates.8,12 In the present work, we report the steady-state and time-resolved studies of single microcrystals of DBPI performed by conventional and confocal fluorescence microscopy showing the effectiveness and limitations of the confocal microscopy on the photodynamic studies of microcrystalline dyes. 2. Experimental Section DBPI (Aldrich, 97%) was used as received. For the measurements of steady-state emission spectra, a CW Ar+ laser (Spectra Physics model 2016) with a single line at 514.5, 488, or 476.5 nm was used to excite the sample (microcrystals sitting on a slide glass) mounted on the stage of a Nikon XF-EFD epi-illumination fluorescence microscope. The size of a microcrystal was measured ((1 µm) with the microscope by using an objective micrometer. Fluorescence emission spectra were recorded by using the above-mentioned microscope by focusing the fluorescence from a single microcrystal through a sharp cutoff glass filter (>490 nm) on the entrance slit of a f/4 monochromator (Ritsu Oyo Kogaku Co. Ltd. model MC 25N) and a photomultiplier (Hamamatsu Photonics R928) setup.7,8,13 © 1997 American Chemical Society
Fluorescence Decay of DBPI Crystals
Figure 1. Fluorescence emission spectra of crystals of DBPI (λexc ) 476.5 nm) with cylindrical shape of 1 µm diameter and 10 µm long (a), 2 µm diameter and 15 µm long (b), 3 µm diameter and 15 µm long (c), and a flat crystal of size 10 × 10 µm (d).
Decay-time measurements were performed by using the timecorrelated single-photon counting technique13 with either a conventional fluorescence microscope or a home-made confocal fluorescence microscope system fabricated recently by the optics supplied by Nikon and Sigma Koki. The fluorescence was filtered with a sharp cutoff glass filter and interference filter and was collected by a Hamamatsu R928 photomultiplier tube.13 The excitation light source was the mode-locked Ar+ ion laser operated at a frequency of 80 MHz. The fluorescence decays measured, therefore, assume sawtooth-like shapes when the emission does not decay practically to zero within the interval of two adjacent pulses (12.5 ns). The fluorescence decay curves thus obtained were analyzed by the full-fit deconvolution method developed in our laboratory.14 The magic angle for our system was determined beforehand by using a thin liquid film of a dilute ethylene glycol solution of rhodamine 6G in the same manner as described in a previous paper15 and was fixed at the same position when a microcrystalline sample was studied since we could not know how the crystalline sample modifies the magic angle thus preset. All the measurements were carried out at room temperature. Time-resolved emission spectra of single microcrystals were recorded directly with the help of a TAC (Ortec model 467) and the f/4 monochromator. Unless otherwise stated, approximately the whole sample microcrystal was irradiated. 3. Results and Discussion 3.1. Absorption and Fluorescence Emission Spectra of Crystalline DBPI. The crystals of DBPI obtained commercially posses a distribution in size and shape. We have classified these into two kinds: one with a cylindrical or stick shape (1-3 µm in diameter and 10-30 µm long) and the other with a flat shape and an arbitrary size of several micrometers. The thickness of flat-shaped microcrystals was estimated to be of the order of a few micrometers visually as it was not possible to measure it precisely with a two-dimensional view of the microscope. Laboratory-prepared crystal samples were in the form of a stick shape and powder that contains crystals of dimensions of a few hundred nanometers. Figure 1 shows the fluorescence emission spectra of single microcrystals of DBPI of different shape. The emission spectrum for a stick-shaped ∼1 µm diameter crystal (spectrum a) shows two prominent bands with peaks located at approximately 575 and 615 nm. Also shown in the figure are the emission spectra for the stick-shaped crystals with 2 and 3 µm diameter and the 10 µm thick crystal. It can be seen that
J. Phys. Chem. B, Vol. 101, No. 41, 1997 8055
Figure 2. Absorption (solid lines) and fluorescence emission (broken lines) spectra of DBPI in chloroform (a) and (c) and a crystalline thin film (b) and (d) (λexc ) 470 nm).
on increasing the diameter of the microcrystal the fluorescence emission intensity of the band at 575 nm decreases relative to the other band. An increase in the emission intensity of the band at 615 nm is observed as a function of the thickness of crystals along with an appearance of a shoulder at 650 nm. For a thicker crystal, the emission spectrum shifts to the red significantly and shows a broad band with a maximum intensity at 630 nm along with a peak at 575 nm and a shoulder at 680 nm (spectrum d). For other crystals of middle size, the emission spectra exhibit shapes varying between those shown for spectra a and d. The length of stick-shaped crystals does not have any notable effect on the spectral shape provided that only the emission from the small irradiated spot is recorded. A preliminary knowledge of the absorption by the microcrystals can be obtained from the absorption spectrum (Figure 2, spectrum b) of the thin film of the crystalline DBPI prepared by depositing sublimated DBPI onto the inner surface of a quartz cuvette in vacuum. The peak positions of the absorption spectrum of the crystalline film thus prepared are approximately the same as those of DBPI in chloroform (20 mM) (Figure 2, spectrum a), but the absorption does not vanish toward the red edge sharply and actually extends up to 650 nm. This tail of the absorption spectrum was checked for dependence on the film thickness by recording absorption spectra at various places of the film of varying thickness through a very small observation area (∼1 mm2). It was confirmed that the extended tail of the spectrum was real. It is interesting to note that a similar absorption spectrum has been reported for a much thinner film of DBPI recently.15 From Figures 1 and 2 it can be seen that the Stokes shift observed for DBPI in the case of stick-shaped microcrystals is larger (∼1500 cm-1) as compared with that in chloroform (450 cm-1). However, the emission spectra of the stick-shaped microcrystals show similarity in shape to that observed for its chloroform solution. Also the vibrational separation in fluorescence for the first two peaks (1160 cm-1) is similar to those observed in chloroform (1130 cm-1) or polystyrene (1180 cm-1). The peak positions of the absorption spectra remain unchanged in solution and crystalline state. Therefore, the apparently larger Stokes shift observed for the microcrystals may arise as a result of the cascade energy transfer to the lower energy sites of DBPI formed in the crystal, which is reasonably inferred from an extended tail in the absorption spectrum (spectrum a in Figure 2). The fluorescence emission spectrum of the crystalline film has a broad band with a maximum located approximately at 640 nm (see Figure 2). 3.2. Time-Resolved Emission. 3.2.1. Fluorescence Decay. The fluorescence decay of microcrystalline DBPI is nonexpo-
8056 J. Phys. Chem. B, Vol. 101, No. 41, 1997
Bisht et al.
Figure 3. Fluorescence decays of a stick-shaped crystal of 1 µm in diameter and 10 µm in length (panel A) observed with an interference filter centered at 558 nm (a), with a cutoff glass filter >560 nm (b), and with a cutoff glass filter >650 nm (c). Panel B shows the fluorescence decays of a flat-shaped (10 × 16 µm) crystal observed with an interference filter centered at 558 nm (e), with a cutoff glass filter >560 nm (f), and with a cutoff glass filter >650 nm (g). Excitation wavelength ) 476.5 nm.
TABLE 1: Fluorescence Lifetimesa and Preexponential Factorsb of Microcrystals of DBPI Obtained as a Result of Triple Exponential Fitting of the Decay Curves Recorded at Different Monitoring Wavelengths (λexc ) 476.5 nm) component lifetimes/ns preexponential factors emission τ2 τ3 R1 R2 R3 wavelength/nm τ1
χ2
〈558 nm〉 >560 nm >650 nm
0.11 0.17 0.17
Size: 1 × 10 µm 0.52 3.65 0.910 0.080 0.004 1.07 0.93 5.39 0.852 0.120 0.030 1.19 1.15 6.56 0.587 0.260 0.160 1.07
〈558 nm〉 >560 nm >650 nm
0.14 0.17 0.18
Size: 10 × 16 µm 0.75 5.17 0.900 0.070 0.030 1.02 1.78 8.88 0.460 0.370 0.170 1.04 1.89 9.62 0.220 0.440 0.340 1.09
a
(0.05 ns. b Normalized to unity.
nential with a large variation in the values of the decay times and preexponential factors depending upon the crystal shape and detection wavelength. Analogous to the fluorescence emission spectra, we have divided the observed fluorescence decay features into two classes: one from the crystals of a cylindrical (or stick) shape with 1-2 µm diameter and the other from flat crystals of a size of several micrometers. Figure 3 (panel A) shows the fluorescence decays of a DBPI crystal of ∼1 µm in diameter and 10 µm in length (1 × 10 µm) as a function of emission wavelength. The measured fluorescence decays are largely nonexponential and show a good fit with a triple-exponential function. The dependence of the fluorescence decay on the emission wavelength of a larger flat crystal of 10 × 16 µm is shown in panel B. Table 1 lists the fluorescence lifetimes and their amplitudes obtained for these two crystals at three different wavelengths. The cylindrical crystal exhibits a faster decay as compared with those for the flat crystal, even when measured at the blue edge of the emission spectrum by collecting emission from the irradiated spot. Two possibilities can be considered for the longer-lived and wavelength-dependent fluorescence lifetime, viz. the selfabsorption effect and the dimer or excimer formation. There exists a considerable overlap between the emission and the absorption spectra of DBPI in solution. The ratio of the emission peaks of DBPI is extremely sensitive to the concentration of DBPI. With increasing the concentration, the emission spectrum can be highly distorted due to the self-absorption. Also
Figure 4. Confocally recorded fluorescence decay of 1 µm diameter (length ) 10 µm) stick-shaped crystal for the emission through the interference filter centered at 558 nm (a) and with a cutoff glass filter >620 nm (b) (λexc ) 514.5 nm). The best-fitted parameters are τ1 ) 0.14 ns, R1 ) 0.98; τ2 ) 0.94 ns, R2 ) 0.02; χ2 ) 1.48 for curve a, and τ1 ) 0.14 ns, R1 ) 0.85; τ2 ) 1.05 ns, R2 ) 0.12; τ3 ) 6.13 ns, R3 ) 0.03, χ2 ) 1.07 for curve b.
the fluorescence decay time and the emission spectra of DBPI are strongly influenced by the self-absorption when measured in a conventional cuvette of 1 cm path length at high concentrations.11 Under crystalline conditions the overlap between the absorption and fluorescence emission spectra becomes much more significant (see Figure 2) because of higher molecular density. A model and general discussion on how the decay times are lengthened as the reabsorption becomes more significant is given in one of our previous papers.16 The dimer or excimer formation does not seem to play a major role for DBPI in solution since the emission spectrum and the decay time of DBPI (3.7 ns) measured in highly concentrated solutions (∼10-3 M in chloroform) in a thin capillary of ∼100 µm diameter (free from the self-absorption) remain unchanged as compared to their dilute solution counterparts. The fluorescence decay times for the crystals, on the other hand, increase with increasing the emission wavelength suggesting that the self-absorption effect is contributing. This is consistent with the observation that the decay times observed for larger crystals are longer and are less sensitive to the emission wavelength as compared with the stick-shaped crystals. The longest decay time of the flat-shaped crystal reaches approximately 9.6 ns on monitoring emission at the red end of the emission spectrum (Figure 3, curve f). With respect to this, it is worth mentioning that the electroluminescence reported recently17 for a ultrathin film (∼10 nm) of DBPI is surprisingly close to the fluorescence spectra given here. 3.2.2. Fluorescence Decay Measured with Confocal Optics. Figure 4 shows the fluorescence decay curve (curve a) of the 1 × 10 µm stick-shaped crystal recorded through an interference filter (λmax ) 558 nm, band pass ) 10 nm) by using the confocal fluorescence microscope upon excitation at 514.5 nm. The fluorescence decay becomes much faster than that recorded with the conventional fluorescence microscope at the same detection wavelengths (see Figure 3). The fluorescence decay tends to lose the nonexponential character as compared to that recorded with the conventional fluorescence microscope. It appears that even the spatial resolution of 0.5 µm is not high enough to remove the self-absorption effects completely. This is so as the fluorescence decay again shows a lengthened component on increasing the detection wavelength. Such an effect clearly indicates that the excitation and the collection of the fluorescence with a limited spatial resolution are actually contributing to the lengthening of the fluorescence lifetime. The decay times obtained from the analysis of the decay curves recorded with the confocal optics at the excitation wavelengths of 476.5 and
Fluorescence Decay of DBPI Crystals
J. Phys. Chem. B, Vol. 101, No. 41, 1997 8057
Figure 5. Photograph of ∼2 × 10 µm cylindrical microcrystal upon exciting with the 475.6 nm line of an Ar+ ion laser. The photograph was taken with a sharp cutoff glass filter (>500 nm) with an exposure time of 1 s. The laser spot is bright with yellow fluorescence, while red fluorescence is visible at both ends of the microcrystal.
515.4 nm are approximately identical and, hence, are independent of the excitation wavelength. This suggests that slight changes in the wavelength of excitation within the absorption band do not lead to a change in the self-absorption effect. In order to visualize the effect of the self-absorption, Figure 5 shows a photograph of a ∼2 × 10 µm crystal taken by exciting it with a focused spot. It can be seen that the crystal shines yellow at the excitation spot but red emission is seen at both edges. The crystal does not appear fluorescent in between the excitation point and the edges. This shows clearly the contribution of the self-absorption effect as well as the optical wave guide nature of the stick-shaped microcrystals. The appearance of the red fluorescence from DBPI, however, has previously been attributed to the excimer formation by the study of DBPI crystals adsorbed on filter paper.10 As we have shown here, a similar red fluorescence can also be observed if the self-absorption effects are not removed properly. Experiments with a thin crystalline film of varying thickness show that the apparent fluorescence decay time increases with the thickness. The stick-shaped crystals, which do not show the broad and red-shifted emission, also exhibit a lengthened component of the fluorescence lifetime beyond 3.7 ns ()radiative fluorescence lifetime of DBPI in solution).11 The fact that the decay profiles are always nonexponential and the emission spectra and the decay curves are strongly dependent upon the thickness of the crystals favors the existence of the selfabsorption. 3.2.3. Time-ResolVed Emission Spectra (TRES). The behavior observed in the emission spectra and fluorescence lifetimes of the microcrystals should also be reflected in the emission spectra of a single microcrystal taken with different time windows if the observed longer lifetimes are due to the self-absorption. While the TRES corresponding to a shorter time should match with the fluorescence emission spectra of the stick-shaped crystals, a time-delayed spectrum should be similar in shape to those obtained from the thick microcrystals having a significant contribution of the self-absorption. We selected a medium sized crystal to have sufficient signal-tonoise ratio for the measurements. Figure 6 shows the TRES for a ∼3 × 10 µm crystal with “early” (0-0.5 ns) and “late” (2.5-10 ns) time windows. The spectra were measured with
Figure 6. TRES for a microcrystal with a diameter of 3 µm (length ) 10 µm) with a time window of 0-0.5 ns (a) and 2.5-10 ns (b) (λexc ) 476.5 nm). The spectra were recorded by using a sharp cutoff glass filter (>500 nm).
wavelength step intervals of 0.5 nm at the rate of 10 s/step. The signal-to-noise ratio was small for the late time window, which resulted in some noise fluctuations built upon the spectrum. The spectra are normalized at 570 nm for convenience. It can be seen that the emission spectrum of the microcrystal having a small contribution from the self-absorption (see Figure 1) changes significantly for the late time window as compared to that measured for the early gate. The spectrum for the late window closely resembles that observed for a thick crystal. 3.2.4. Fast Fluorescence Decay of the Microcrystals. The fast decay time (0.11-0.14 ns) for DBPI under microcrystalline conditions is puzzling. Several possibilities such as the energy migration or hopping followed by quenching by some trap sites inside the microcrystals, quenching by oxygen, or contribution from the Mie resonances,18 can be considered to account for the observed nonexponential nature of the decay curves with a fast decaying component. An extensive study on the concentration dependence of the fluorescence lifetime in solution indicates that DBPI does not show concentration quenching up to the concentration of 20 mM but is quenched by dissolved oxygen nearly at the rate of diffusion.11 In order to find out the cause for the short lifetime of the microcrystals, it was essential to examine the effect of quenching by oxygen, if any, by measuring the decay from the crystal under deaerated conditions. The thin
8058 J. Phys. Chem. B, Vol. 101, No. 41, 1997 crystalline film of varying thickness deposited onto the surface of the wall of a quartz cuvette as mentioned earlier was used for such a study. The cuvette was kept sealed for the measurements under deaerated conditions. The measured fluorescence decays by using the confocal optics were nonexponential with the presence of a fast decay component and were not affected by the presence of oxygen. This indicates that the fast fluorescence decay is not due to the quenching of emission by oxygen in the air. A crude measurement of the refractive index of the DBPI crystal gave a value of 1.76.19 The radiative lifetime for the DBPI crystal predicted by its dependence on refractive index is approximately 2.5 ns (1.452/1.762 × 3.7 ns ) 2.5 ns). Therefore, a significant shortening in the decay time (3.5 ns) accompanied by a relative increase in the emission at longer wavelengths of the DBPI microcrystal are attributed to the self-absorption effects observed even at the level of a few micrometers. This has been confirmed through the measurements of the fluorescence decay by resolving a very
Bisht et al. small space in the single microcrystal by using the confocal fluorescence microscope. The present study shows that even under sizes of as small as 1 µm care has to be taken against the artifact of self-absorption when calculating the rate parameters. Excimer formation may have contribution under certain crystal structures, but, as yet, there is no clear cut proof for any excimer emission or a new emitting species in DBPI under microcrystalline conditions or in bulk solutions. Absence of any complication such as photodecomposition, excimer formation, or concentration quenching makes DBPI most suitable for the study of photodynamics in the crystalline state. Acknowledgment. P.B.B. thanks Japan Society for the Promotion of Science (JSPS) for financial support during his stay at KIT. References and Notes (1) (a) Betzig, F.; Chichester, R. J. Science 1993, 262, 1422. (b) Kino, G. S.; Corle, T. R. Phys. Today 1989, 42 (Sept), 55. (2) Vander Bout, D. A.; Kerimo, J.; Higgins, D. A.; Barbara, P. F. J. Phys. Chem. 1996, 100, 11843. (3) Reid, P. J.; Higgins, D. A.; Barbara, P. F. J. Phys. Chem. 1996, 100, 3892. (4) Higgins, D. A.; Reid, P. J.; Barbara, P. F. J. Phys. Chem. 1996, 100, 1174. (5) Dunn, R. C.; Holtom, G. R.; Mets, L.; Xie, X. S. J. Phys. Chem. 1994, 98, 3094. (6) (a) Smith, D. A. McL.; Williams, S. A.; Miller, R. D.; Hochstrasser, R. M. J. Fluoresc. 1994, 4, 137. (b) Trautman, J. K.; Macklia, J. J. Chem. Phys. 1996, 205, 221. (7) Bisht, P. B.; Fukuda, K.; Hirayama, S. Chem. Phys. Lett. 1996, 258, 71. (8) Bisht, P. B.; Fukuda, K.; Hirayama, S. J. Chem. Phys. 1996, 105, 9349. (9) Rademacher, A.; Ma¨rkle, S.; Langhlas, H. Chem. Ber. 1982, 115, 292. (10) Ford, W. E.; Kamat, P. V. J. Phys. Chem. 1987, 91, 6373. (11) El-Daly, S. A.; Okamoto, M.; Hirayama, S. J. Photochem. Photobiol. A 1995, 91, 105. (12) Bisht, P. B.; Fukuda, K.; Hirayama, S. To be published. (13) Hirayama, S. In Progress in Photochemistry and Photophysics; Rabek, J. F., Ed.; CRC Press: Boca Raton, FL, 1992, Vol. 6, p 1. (14) Sakai, Y.; Hirayama, S. J. Luminesc. 1988, 39, 145. (15) Pandey, K. K.; Hirayama, S. J. Photochem. Photobiol. A 1996, 99, 165. (16) Sakai, Y.; Kawahigashi, M.; Minami, T.; Inoue, T.; Hirayama, S. J. Luminesc. 1989, 42, 317. (17) Yoshida, M.; Fujii, A.; Ohmori, Y.; Yoshino, K. Jpn. J. Appl. Phys. 1996, 35, L397. (18) Bohren, C. F.; Huffman, D. R. Absorption and scattering of light by small particles; John Wiley & Sons, New York, 1983. (19) The refractive index was measured by Fission-Track Co. Ltd., Kyoto.
