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Singlet Oxygen Images of Heterogeneous Samples: Examining the Effect of Singlet Oxygen Diffusion across the Interfacial Boundary in Phase-Separated Liquids and Polymers Ingo Zebger, Lars Poulsen, Zhan Gao, Lars Klembt Andersen, and Peter R. Ogilby* Department of Chemistry, University of Aarhus, Langelandsgade 140, DK-8000 A° rhus, Denmark Received April 7, 2003. In Final Form: May 28, 2003 Optical images of glassy, phase-separated polymer films have been generated using the 1270 nm phosphorescence of singlet molecular oxygen. Specifically, upon irradiation of immiscible blends of polystyrene and poly(ethylene-co-norbornene) that contain a singlet oxygen sensitizer, phase-separated droplets as small as ∼10 µm in diameter could be resolved using a microscope designed to detect singlet oxygen phosphorescence. This study demonstrates that it is possible to create singlet oxygen images of systems in which the sensitizer is not mobile (i.e., systems in which the effects of sensitizer bleaching cannot be rectified by diffusion of more dye into a given volume). The effect of singlet oxygen diffusion across the interfacial boundary between phase-separated domains of both liquid and polymer samples has also been examined. For singlet oxygen created in one phase, diffusion into a second phase in which the quantum yield of singlet oxygen phosphorescence is larger and oxygen is more soluble gives rise to a significant change in the intensity of the singlet oxygen signal at the interface. This effect can be pronounced when a long singlet oxygen lifetime facilitates singlet oxygen diffusion over large distances, but can be mitigated when interfacial tension yields a phase boundary with appreciable curvature. Boundary curvature in thin, phase-separated films of polystyrene/poly(ethylene-co-norbornene) is slight. Moreover, the singlet oxygen lifetime in these polymers is sufficiently short that, within its lifetime, singlet oxygen cannot diffuse over an appreciable distance. Under such conditions, singlet oxygen images of interfacial boundaries are sufficiently sharp as to make this optical technique useful for a range of fundamental studies.
Introduction 1
Singlet molecular oxygen (a ∆g) is an important intermediate in many chemical and biological processes, in part, because of its unique reactivity that can result in the oxidation of organic molecules.1 Indeed, singlet oxygen plays significant roles in processes that range from the degradation of polymeric materials2 to events that result in both the death as well as protection of biological cells.3-5 Because many such systems of interest are inherently heterogeneous, it is of great interest to directly monitor singlet oxygen with spatial resolution and, in turn, ultimately create a singlet oxygen image of the system under study. We have embarked on a multifaceted program to develop optical microscopes capable of directly detecting singlet oxygen. We recently established that, upon steady-state irradiation of a sensitizer, singlet oxygen can be detected with a lateral resolution of ∼2.5 µm via the weak O2(a1∆g) f O2(X3Σg-) phosphorescent transition at ∼1270 nm.6 In * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Foote, C. S.; Clennan, E. L. In Active Oxygen in Chemistry; Foote, C. S., Valentine, J. S., Greenberg, A., Liebman, J. F., Eds.; Chapman and Hall: London, 1995; pp 105-140. (2) Scurlock, R. D.; Wang, B.; Ogilby, P. R.; Sheats, J. R.; Clough, R. L. J. Am. Chem. Soc. 1995, 117, 10194-10202. (3) Weishaupt, K. R.; Gomer, C. J.; Dougherty, T. J. Cancer Res. 1976, 36, 2326-2329. (4) Hideg, E.; Ogawa, K.; Ka´lai, T.; Hideg, K. Physiol. Plant. 2001, 112, 10-14. (5) Chen, W. L.; Xing, D.; Tan, S.; Tang, Y.-H.; He, Y. Luminescence 2003, 18, 37-41. (6) Andersen, L. K.; Gao, Z.; Ogilby, P. R.; Poulsen, L.; Zebger, I. J. Phys. Chem. A 2002, 106, 8488-8490.
turn, singlet oxygen images of heterogeneous samples were created using this luminescent signal.6 A lateral resolution of 2.5 µm is close to the diffraction limit for light at 1270 nm, which, depending on the optics used, is ∼ 1.5-2.0 µm. In this study, experiments were performed on phaseseparated mixtures of toluene and water. Specifically, singlet oxygen was produced upon irradiation of the sensitizer 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP) dissolved in the toluene phase. Because TPP is not soluble in water, we were able to distinguish domains that contained singlet oxygen from those that did not. There are several significant features of this liquid system used in our initial study. First, the sensitizers could diffuse during the course of the experiment in which the singlet oxygen image was constructed. Thus, for samples involving relatively large toluene domains, the adverse effects of photoinduced sensitizer bleaching were mitigated simply because sensitizers were constantly moving in and out of the irradiated area. For samples in which sensitizer exchange was limited, however, it was difficult to obtain acceptable singlet oxygen images due to irreversible reactions that depleted the sensitizer and/ or gave rise to products whose luminescence interfered with that of singlet oxygen. Second, in liquid systems where solute diffusion is pronounced, intermolecular quenching by oxygen plays a significant role in the deactivation of the sensitizer excited states. Under such conditions, the ratio of the singlet oxygen yield to the yield of sensitizer luminescence can be quite large. In systems where solute diffusion is restricted, however, one can have the added complication of more intense luminescence from
10.1021/la0301487 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/26/2003
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the sensitizer that can interfere with the desired singlet oxygen signal. For the present study, we set out to establish if it is feasible to create singlet oxygen images from rigid samples in which the sensitizer is not mobile. We now report that singlet oxygen images of such systems can indeed be created. These data, along with the results of a study on the effects of singlet oxygen diffusion across interfacial boundaries, indicate that it is now reasonable to consider singlet oxygen imaging studies that involve sensitizers enclosed in small vesicles as well as sensitizers immobilized in biological cells. Experimental Section Details of the instrumentation and approach used in the present study are described elsewhere.6 Briefly, experiments were performed using a modified Olympus inverted microscope (model IX70) equipped with a custom-made InGaAs linear array detector that had 512 elements, each of which was 50 µm × 50 µm (Princeton Instruments/Roper). The sensitizer was irradiated using the output of a 75 W steady-state Xe lamp that was coupled onto the sample through the microscope objective using a dichroic mirror. Luminescence from the sample was collected by the microscope objective and transmitted through this same dichroic mirror onto the InGaAs detector, which was operated at -100 °C. The 1270 nm phosphorescence of singlet oxygen was spectrally isolated using an interference filter (Barr Associates). When using a linear array detector in such a microscope, one obtains the luminescence intensity profile of a “slice” across the sample. Two-dimensional images are obtained by laterally translating the sample on the microscope stage and thus recording successive slices. Along the axis of the detector array (x-axis), the spatial resolution available is determined by the combination of the element size in the detector array and the objective magnification. For the current experiments, a 20× objective was used (numerical aperture of 0.4) that, in turn, yields a lateral resolution of 2.5 µm per pixel. Note that, in making this statement, we are not considering ramifications of the Nyquist sampling theorem7,8 which, when applied to our conditions, means that we would only be able to provide a faithful image of objects larger than 5 µm. On the other hand, when using this detector array, we are indeed able to resolve a 10 µm long object using ∼4 pixels (Figure 1). Along the scanning axis (y-axis), the accuracy of the translation stage and the size of each step in which the sample is moved also contribute to the spatial resolution. For the current experiments, a step size of 2.5 µm was used. For the work reported herein, several significant modifications were made to the microscope: (1) The breadth of the spectral output of the Xe lamp used to irradiate the sensitizer was significantly reduced. As in our first study,6 a 2 cm path length water filter, KG-2 and KG-1 filters (Schott), and a hot mirror (CVI Inc.) removed the infrared radiation and limited the incident light to the range 400-700 nm. An interference filter with a band-pass centered at 400 nm and a bandwidth at half-maximum of ∼70 nm (Corion, Inc.) further reduced the spectral range to a narrow band that coincided with an absorption band of the sensitizers used. (2) A computer-controlled shutter was used to limit the extent to which the sample was exposed to the output of the Xe lamp. Specifically, the shutter was opened only during the period in which data were being collected by the InGaAs array. (3) The gain of the analog amplifier at the analog-to-digital converter used for signal processing was increased. This reduced the sampling time necessary to achieve an acceptable signal level, which, in turn, made it possible to significantly reduce the time for which the sample was exposed to the output of the Xe lamp. (4) The sample was mounted on a computer-controlled, screwdriven x-y stage (0.1 µm resolution) with associated linear encoders to verify the position of the sample (Physik Instrumente model M-126CG). Images of the sample could thus be more rapidly (7) Pawley, J. B. In Handbook of Biological Confocal Microscopy, 2nd ed.; Pawley, J. B., Ed.; Plenum Press: New York, 1995; pp 19-37. (8) Webb, R. H.; Dorey, C. K. In Handbook of Biological Confocal Microscopy, 2nd ed.; Pawley, J. B., Ed.; Plenum Press: New York, 1995; pp 55-67.
Figure 1. (A) Visible image of a lithographic grating. The dark stripes are ∼10 µm wide. (B) Plot of the intensity of 1270 nm light transmitted by the grating in part A against individual pixels in the InGaAs array. Data were recorded such that the array collected information from a “slice” across the grating, and the response of each pixel is marked with an “×”. For this test, the grating was illuminated from the backside using a tungsten lamp. Note that the dark stripes are resolved with ∼4 pixels, which is consistent with a resolution of ∼2.5 µm/pixel. The data indicate that the transmittance through the slightly wider, lighter stripes varies with position. constructed simply because data from the respective sample “slices” could be more efficiently collected. The poly(ethylene-co-norbornene) used in this study was obtained from Hoechst Ticona, Germany. In the manufacturer’s system of nomenclature, the materials used were TOPAS 8007 (Mw ) 116 000 g mol-1, Tg ) 78.8 °C) and TOPAS 6013 (Mw ) 102 000 g mol-1, Tg ) 140 °C), which are comprised of 35 and 51 mol % norbornene, respectively.9 The material was obtained in the form of pellets and was used without further purification. Polystyrene, Aldrich, (Mw ) 280 000 g mol-1, Tg ∼ 100 °C) was likewise obtained as pellets and used without further purification. Films of TOPAS 6013 were prepared by spin casting. In a typical procedure, the polymer pellets and the singlet oxygen sensitizer were dissolved in benzene. The amount of polymer used was generally 20 wt %. These benzene solutions were maintained at 60 °C for 2 h to ensure polymer dissolution, and then they were spin-cast onto glass plates that were likewise maintained at ∼60 °C. A Headway Research model EC101DTR790 photoresist spinner was used, and a typical spinning speed was ∼4000 rpm. Films obtained from a blend of polystyrene and poly(ethylene-co-norbornene) were obtained by solution casting without spinning. Once again, the polymers and the sensitizer were simply dissolved in benzene and then cast onto a microscope slide. The overall amount of both polymers in the benzene solution was 5 wt %. To obtain phase separated samples that best suited our needs, films were prepared using polystyrene and TOPAS 8007 in the relative ratios of either 50/50 or 20/80 wt %, respectively. Once prepared, the films were allowed to stand for 24 h at ambient temperature and pressure, and then they were subsequently placed under vacuum for an additional 24 h. Finally, the films were annealed at 50 °C for 24 h under vacuum using a Lab-Line Duo-Vac oven (model 3620-ST-1). Under these conditions, films with a thickness in the range ∼10-25 µm were (9) Poulsen, L.; Zebger, I.; Klinger, M.; Eldrup, M.; Sommer-Larsen, P.; Ogilby, P. R. Submitted for publication.
Singlet Oxygen Images of Heterogeneous Samples
Figure 2. Images of a scratch in a film of poly(ethylene-conorbornene) containing the singlet oxygen sensitizer lipo-TPP. (A) A transmission-based image of the sample in the visible region of the spectrum. (B) Image of the same region shown in part A constructed using the 1270 nm phosphorescence of singlet oxygen. The latter image was assembled using 132 linear array “slices”, each of which contained data obtained in a 20 s exposure time. The colors chosen for the singlet oxygen image are arbitrary and reflect the intensity scale used. generally produced. The concentration of sensitizer in the film was typically 1.5 × 10-3 M. The sensitizers used in this study were meso-tetraphenylporphine, TPP (Aldrich), and tetrakis(4tetradecyloxymethylphenyl)porphine, so-called lipo-TPP (Porphyrin Systems, Germany). Both compounds were used as received. TPP is readily soluble in polystyrene but is only sparingly soluble in poly(ethylene-co-norbornene). Lipo-TPP is soluble in poly(ethylene-co-norbornene). For the experiments performed in mixtures of CS2 and D2O where singlet oxygen was created in the D2O phase, the sensitizer used was TPyP [5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H,23H-porphine tetra-p-tosylate salt], obtained from Aldrich and used as received. For experiments in which singlet oxygen was created in the CS2 phase, TPP was the sensitizer. D2O was obtained from Deutero GmbH (99.9% D), and CS2, from Aldrich (spectroscopic grade). These liquid-phase experiments were performed in a 100 µm path length cell with sensitizer concentrations of ∼3 × 10-4 M.
Results and Discussion Image of a Scratch in a Glassy Polymer Film. In an initial test of our ability to create singlet oxygen images of samples in which the sensitizer is immobilized, films of a glassy polymer (TOPAS 6013) containing the sensitizer lipo-TPP were cast onto a microscope slide. The polymer film was then scratched with a scalpel such that a channel void of the polymer was created. When making this scratch, material from inside the channel was pushed up onto the sides, creating domains in which the thickness of the polymer varied. Images of this scratch in the polymer film are shown in Figure 2. Scratch-induced differences in the thickness of the polymer are clearly evident in the singlet oxygen intensities observed. Most importantly, for data recorded using the conditions described in the Experimental Section, the sensitizer was stable over a prolonged period
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Figure 3. Plot of the light intensity detected by the InGaAs array against the individual pixels in the array. Data were recorded such that the array collected information from a slice across the scratch shown in Figure 2. (A) Data recorded from a given sample position both before and after the sensitizer had been irradiated for 45 min. (B) Data recorded using a narrow band interference filter centered at 1200 nm, demonstrating the absence of background luminescence that could interfere with the singlet oxygen signal recorded at 1270 nm. A correction to account for inhomogeneities in the Xe lamp (a flat-field correction) was not applied to the data.
of irradiation and interference from near-IR background luminescence did not complicate the detection of the singlet oxygen signal. These points are illustrated in Figure 3, in which data from a single slice across the scratch are shown. In Figure 3A, we demonstrate that the intensity profile of a given slice across the scratch does not vary appreciably over the time required to scan the image. (Actually, prior to recording the data shown in Figures 2 and 3, this particular sample had already been exposed to light for ∼2 h during the course of preliminary experiments.) In Figure 3B, we demonstrate the absence of luminescence from other sources that could interfere with the detection of singlet oxygen. This latter experiment relies on the fact that, in both liquids and polymers, the phosphorescence of singlet oxygen occurs in a discrete narrow band centered at ∼1270 nm.10,11 On the other hand, “background” luminescence generally derives from broadband tails of fluorescent and/or phosphorescent transitions whose origins are in the visible region of the spectrum. The most prominent source of such luminescence is generally the singlet oxygen sensitizer itself. Thus, if one simply replaces the 1270 nm interference filter designed to isolate singlet oxygen phosphorescence with a corresponding narrow band interference filter centered at 1200 nm, one should (10) Dam, N.; Keszthelyi, T.; Andersen, L. K.; Mikkelsen, K. V.; Ogilby, P. R. J. Phys. Chem. A 2002, 106, 5263-5270. (11) Wessels, J. M.; Rodgers, M. A. J. J. Phys. Chem. 1995, 99, 1758617592.
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Figure 4. Images of a glassy, phase-separated blend of poly(ethylene-co-norbornene) droplets in a polystyrene matrix that contains the singlet oxygen sensitizer TPP. (A) A transmission-based image of the sample in the visible region of the spectrum. (B and C) Images of the same region shown in part A constructed using the 1270 nm phosphorescence of singlet oxygen (features of the singlet oxygen image are better observed if the data are plotted using different color schemes to represent the signal intensity). In parts B and C, the spots identify poly(ethylene-co-norbornene) droplets in which there is very little, or no, singlet oxygen. The images were constructed using 132 linear array “slices”, each of which contained data obtained in a 30 s exposure time. The vertical lines in the singlet oxygen images derive from inhomogeneities in the intensity profile of the Xe lamp as well as from differences in the gain of respective elements in the detector array (i.e., “hot” pixels).
totally discriminate against the singlet oxygen signal but have minimal effect on the background signal. The data shown in Figure 3B, in which interfering background luminescence is conspicuously absent, were recorded upon irradiation of the sensitizer using a narrow spectral band centered at ∼400 nm (see Experimental Section). On the other hand, when the poly(ethylene-conorbornene) samples containing lipo-TPP were irradiated using a large spectral bandwidth (∼400-700 nm), a comparatively intense near-IR background signal was observed. These experiments suggest that the interfering near-IR luminescence could arise, for example, from impurities in the sample that absorb at longer wavelengths than does the principal transition in lipo-TPP. In the present context, however, the important point is that interfering luminescence can be significantly reduced, and in some cases totally avoided, simply by irradiating the sensitizer over a narrow spectral bandwidth. Data such as those in Figure 3B are also important in substantiating that we indeed are looking at singlet oxygen. Specifically, for all of the experiments reported herein, the luminescence signal assigned to singlet oxygen phosphorescence derives from a distinct transition at 1270 nm. Moreover, upon removing oxygen from the system either by evacuating the sample or by equilibrating the sample with nitrogen gas, this emission at 1270 nm disappears.
Images of Phase-Separated Droplets. In a second test of our ability to create singlet oxygen images of samples in which the sensitizer is immobilized, we examined phase-separated blends of poly(ethylene-conorbornene) and polystyrene containing TPP as the singlet oxygen sensitizer. In a film cast from a blend of polystyrene and TOPAS 8007, for example, distinct phase-separated droplets of TOPAS 8007 are formed in polystyrene (Figure 4). An advantage of such a sample, certainly for the experiments in this study, is the range of drop sizes formed; the largest drops shown in Figure 4 have a diameter of ∼100 µm, and the smallest have a diameter < 1 µm. TPP is only sparingly soluble in poly(ethylene-co-norbornene), and thus, upon irradiation, singlet oxygen is formed almost entirely in the polystyrene phase. The data clearly reveal that, under our experimental conditions, phase-separated domains with a diameter as small as ∼10 µm can readily be resolved in the singlet oxygen image (Figure 4). Singlet Oxygen Diffusion across an Interfacial Boundary. When examining singlet oxygen images such as those shown in Figure 4, it is important to address the issue of how accurately one can resolve the boundary between different phases and to identify factors that influence the phosphorescence intensity observed at an interfacial boundary. Singlet oxygen is a transient species. Once formed, it will decay with a lifetime that can range from nanoseconds
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to tens of milliseconds, depending on the surrounding environment.12 Thus, a given singlet oxygen molecule may diffuse a great distance from where it was produced before decaying. In turn, such diffusion could blur the singlet oxygen image of an interfacial boundary or of a small phase-separated droplet. To address this issue, we thought it best to first model the singlet oxygen concentration profile across a simple one-dimensional interface between two liquids. By using liquids in which the diffusion coefficient of oxygen is comparatively large, we are essentially modeling a “worst case” scenario. Moreover, we can exacerbate this worst case scenario by choosing liquids in which the singlet oxygen lifetime is comparatively long. To this end, we examine a situation where singlet oxygen is produced in D2O and then diffuses across an interface into CS2. The lifetime, τ∆, of singlet oxygen in D2O is ∼65 µs.13 Assuming a typical diffusion coefficient for oxygen, D, in liquids of ∼3 × 10-5 cm2 s-1,14 the average distance traveled by singlet oxygen in D2O in 65 µs is approximately 0.62 µm [root-mean-square linear displacement ∼ (2τ∆D)1/2]. In CS2, when we assume a singlet oxygen lifetime that reflects some quenching by ground-state oxygen as well as the sensitizer, τ∆ ∼ 29 ms,15,16 the root-mean-square linear displacement is ∼12 µm. To model this D2O/CS2 system, we assume singlet oxygen diffusion occurs along the spatial coordinate x from the D2O domain (x < 0), across the interface at x ) 0, and into the CS2 phase (x > 0). Using Fick’s second law of diffusion,17 the steady-state singlet oxygen concentration, c, in D2O can be described using the following differential equation:
∂2c ∂c ) 0 ) D 2 - kdD2Oc + Rexφ∆ ∂t ∂x
(1)
In eq 1, kdD2O is the pseudo-first-order rate constant for singlet oxygen decay in D2O (i.e., τ∆-1) and, thus, when multiplied by c, expresses the rate of singlet oxygen decay. The rate of singlet oxygen formation can be expressed as the product of the rate at which excited-state sensitizer is produced, Rex, (i.e., moles of photons absorbed per unit volume per time) and the quantum yield of singlet oxygen production, φ∆. In the CS2 phase, no singlet oxygen is produced, and the singlet oxygen concentration is given by
∂2c ∂c ) 0 ) D 2 - kdCS2c ∂t ∂x
(2)
where kdCS2 is the rate constant for singlet oxygen decay in CS2. The solutions to these equations18 yield the singlet oxygen concentration profile as a function of distance x from the interface in both D2O and CS2 (eqs 3a and 3b, respectively):
(x )
c(x) ) c0 + k′1 exp x
(x )
c(x) ) k1 exp x
(x ) (x )
kdD2O + k′2 exp -x D
kdCS2 + k2 exp -x D
kdD2O D (3a)
kdCS2 D
(3b)
The constants ki and k′i can be found using the following boundary conditions: (1) The singlet oxygen concentration at x ) -∞ is equal to the steady-state singlet
Figure 5. Calculated singlet oxygen concentration profile across a one-dimensional interface between D2O (x < 0) and CS2 (x > 0). The singlet oxygen concentrations, c, are normalized by the concentration, c0, in the bulk D2O solution.
oxygen concentration in a homogeneous D2O solution, c0 ) Rexφ∆/kdD2O. Thus, k′2 ) 0. (2) The singlet oxygen concentration at x ) +∞ is 0. Thus, k1 ) 0. (3) We assume that, at the interface, both the singlet oxygen partial pressure and the flux are continuous (i.e., cCS2/SCS2 ) cD2O/ SD2O and ∂cCS2/∂x ) ∂cD2O/∂x, respectively, where S is the oxygen solubility in the given solvent). Using eqs 3a and 3b, the given boundary conditions, and a ratio of the oxygen solubilities of SCS2/SD2O ) 5.7,19 the singlet oxygen concentration profile across the D2OCS2 interface was calculated (Figure 5). It is important to again note that, in this model, we stipulate that singlet oxygen is only produced in the D2O phase. The calculations indicate that the singlet oxygen concentration in D2O will decrease near the interface but that the singlet oxygen concentration in CS2 within the first ∼12 µm of the interface will be significantly larger than that in the bulk D2O solution, c0. For an imaging experiment in which singlet oxygen is monitored via its phosphorescence at 1270 nm, it is necessary to consider the analogous phosphorescence intensity profile across the D2O-CS2 interface. In a given solution, the singlet oxygen phosphorescence intensity is proportional to the product of the singlet oxygen concentration and the rate constant for singlet oxygen radiative decay, kr. Since the ratio krCS2/krD2O ∼ 17,20 it is expected that, for the one-dimensional phase boundary modeled in Figure 5, the singlet oxygen phosphorescence intensity within the first few micrometers of the interface should be a factor of ∼80 times larger in CS2 than in D2O. Under our current experimental conditions, in which we have a lateral spatial resolution of ∼2.5 µm, this expected domain of high singlet oxygen intensity in CS2 could be observed. Specifically, upon examining the singlet oxygen intensity profile across the D2O-CS2 interface, under conditions in which singlet oxygen is produced only (12) Wilkinson, F.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref. Data 1995, 24, 663-1021. (13) Ogilby, P. R.; Foote, C. S. J. Am. Chem. Soc. 1982, 104, 20692070. (14) Tsushima, M.; Tokuda, K.; Ohsaka, T. Anal. Chem. 1994, 66, 4551-4556. (15) Opriel, U.; Seikel, K.; Schmidt, R.; Brauer, H.-D. J. Photochem. Photobiol., A: Chem. 1989, 49, 299-309. (16) Schmidt, R.; Afshari, E. Ber. Bunsen-Ges. Phys. Chem. 1992, 96, 788-794. (17) Crank, J. The Mathematics of Diffusion, 2nd ed.; Oxford Press: Oxford, 1975. (18) Arfken, G. B.; Weber, H. J. Mathematical Methods for Physicists, 4th ed.; Academic Press: San Diego, CA, 1995. (19) IUPAC Solubility Data Series. Volume 7: Oxygen and Ozone; Battino, R., Ed.; Pergamon Press: Oxford, 1981. (20) Scurlock, R. D.; Nonell, S.; Braslavsky, S. E.; Ogilby, P. R. J. Phys. Chem. 1995, 99, 3521-3526.
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Figure 6. Illustration of how the curved edge of a phaseseparated droplet would influence the singlet oxygen phosphorescence imaged onto the array detector. For a system in which singlet oxygen is produced solely in the D2O phase, the effects of diffusion across the interface into the CS2 phase (white arrows) would be registered over a large portion of the array detector (dark arrows) simply because the interface itself covers a large portion of the array detector. For a series of vertical slices through the sample, the gradual change in the D2O/CS2 ratio due to the curved interface will mitigate the abrupt change in phosphorescence intensity expected from a planar interface (Figure 5).
in the D2O phase, the first 4-5 pixels recorded from the CS2 phase should show this large discrepancy in intensities. In a real system, however, the boundary between the CS2 and D2O phases will not be as simple as that used in the model calculation. Rather, due to interfacial tension, the boundary will have significant curvature that could extend over tens of micrometers. Thus, the predicted change in singlet oxygen intensity at the interface will be influenced, and potentially mitigated, by the effects of a curved interface. This point is illustrated in Figure 6. To test these hypotheses, we recorded the steady-state singlet oxygen phosphorescence intensity across a variety of phase-separated boundaries. Data from liquid samples were recorded using a 100 µm path length cell, and thus, the effects of droplet curvature could be quite pronounced. Upon examining the intensity profile across the interface between D2O and CS2, we were indeed able to observe a substantial change in the signal intensity. Specifically, for singlet oxygen produced in the D2O phase, the data clearly indicate a significant increase in the singlet oxygen phosphorescence intensity as one progresses from the D2O domain across the phase boundary into the CS2 domain (Figure 7A). Thereafter, as one progresses further into the CS2 domain, the singlet oxygen signal drops to zero, as expected. The mitigating effects of a curved interface are also clearly manifested in the data; in progressing from the D2O domain into the CS2 domain, the increase in singlet oxygen intensity does not occur abruptly as modeled in Figure 5. Rather, the intensity increases gradually over a distance of ∼40-45 µm before dropping to zero. It should also be noted that, for experiments performed using phase-separated liquids, we have observed some lateral translation of the interface boundary during the course of an experiment. This latter effect can become significant when using comparatively long data acquisition times (e.g., 30 s per “slice”, as in Figure 7A). Such translation, or boundary jitter, which may be exacerbated by increases in the sample temperature upon irradiation, will influence the data in the same way as boundary curvature.
Figure 7. Plots of the singlet oxygen phosphorescence intensity recorded by the array detector against the individual pixels in the array. Data were recorded from two different phaseseparated samples of D2O and CS2 positioned such that the array detector cut across a phase boundary. (A) Singlet oxygen was created in the D2O phase (TPyP as sensitizer). The data shown were acquired over a period of 30 s. (B) Singlet oxygen was created in the CS2 phase (TPP as sensitizer). The data shown were acquired over a period of 1 s. A flat field correction was applied to the data in both cases.
To further substantiate that we can indeed accurately predict and detect phenomena that influence the singlet oxygen intensity at a phase boundary, we consider the CS2/D2O solvent pair under conditions in which singlet oxygen is produced in the CS2 domain and then diffuses across the phase boundary into D2O. In this case, because the singlet oxygen lifetime and rate constant for radiative decay as well as the oxygen solubility are all smaller in D2O than in CS2, one simply expects a decrease in the signal intensity at the phase boundary, broadened by the effects of interface curvature. This is indeed observed (Figure 7B). Singlet oxygen phosphorescence intensities recorded across interfaces in the polystyrene/poly(ethylene-conorbornene) films show sharp phase boundaries that are generally less than ∼5 µm, depending on the size of the droplet being imaged (see Figure 4C). In comparison to the data recorded from the CS2/D2O system, this feature of the polymer system derives, in part, from interfacial tension that yields a phase boundary with less curvature. Moreover, with a path length in the range ∼15-25 µm, the polymer samples are much thinner than the liquid samples. Hence, the effect of any interface curvature in the polymer sample will be less pronounced on the singlet oxygen image observed. Of course, even if we had an ideal planar interface in the polymer film, as modeled in Figure 5 for the liquid samples, we would nevertheless not be able to resolve the effects of oxygen diffusion across the interface with our present resolution of ∼2.5 µm/pixel. Given the comparatively small oxygen diffusion coefficient in the glassy polymers and the comparatively short singlet oxygen lifetime in media that contain C-H bonds, singlet
Singlet Oxygen Images of Heterogeneous Samples
oxygen will simply not diffuse very far in its lifetime. For example, in polystyrene where D ) 2 × 10-7 cm2 s-1 and τ∆ ∼ 20 µs,21,22 the mean linear displacement in 20 µs is ∼30 nm. Such samples, in which singlet oxygen images are characterized by comparatively sharp interfacial boundaries, can be used for a number of fundamental studies. For example, using the polystyrene/poly(ethyleneco-norbornene) blend, the effect of heterogeneous domains on oxygen diffusion across a film could be monitored in a series of time-dependent singlet oxygen images. Conclusions We have demonstrated that images of phase-separated materials can be generated using the phosphorescence of singlet molecular oxygen as an optical probe. In particular, upon irradiation of materials that contain an immobile (21) Gao, Y.; Baca, A. M.; Wang, B.; Ogilby, P. R. Macromolecules 1994, 27, 7041-7048. (22) Clough, R. L.; Dillon, M. P.; Iu, K.-K.; Ogilby, P. R. Macromolecules 1989, 22, 3620-3628.
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singlet oxygen sensitizer, phase-separated domains as small as ∼10 µm in diameter were readily resolved. The results reported herein establish that singlet oxygen images of heterogeneous systems can be recorded under conditions pertinent for the study of many chemical and biological processes. Acknowledgment. This work was supported by a grant from the Materials Research Program of the Danish Research Council and by a grant for the “Center for Oxygen Microscopy” from the Danish Natural Science Research Council. L.P.’s Ph.D. stipend was provided by the Danish Polymer Center. The authors thank Markus Klinger for assistance in sample preparation, Dieter Ruchatz of Hoechst Ticona (Germany) for providing the TOPAS used in this study, and P. S. Ramanujam (Risø National Laboratory) for the grating used in Figure 1. LA0301487
