- Email: [email protected]
Efficient excitation energy transfer among multiple dyes in polystyrene microspheres
Journal of Luminescence 79 (1998) 225—231
Efficient excitation energy transfer among multiple dyes in polystyrene microspheres Daniel V. Roberts , Bruce P. Wittmershaus *, Yu-Zhong Zhang, Sharon Swan, Michael P. Klinosky School of Science, Pennsylvania State University: Erie, The Behrend College, Station Rd., Erie, PA 16563-0203, USA Molecular Probes Inc., 4849 Pitchford Ave. Eugene, OR 97402-9144, USA Received 27 December 1997; received in revised form 28 May 1998; accepted 10 July 1998
Abstract Our comparison of fluorescence excitation and absorption spectra from 40 nm polystyrene spheres containing six different dyes illustrates efficient excitation energy transfer among the dyes. The six-dye spheres collective absorption covers the entire visible region. The five dyes with the highest first excited state energy levels transfer their excitations to the sixth dye of the lowest energy level. The average excitation transfer efficiency to the sixth dye is 95% over most of the visible spectrum. As a result of this transfer, the spheres have a fluorescence maximum at 718 nm, the peak of emission from the lowest energy level dye. Each sphere functions as an artificial light-harvesting network. They have practical applications as fluorescent labels for biological systems and act as an efficient, broadly absorbing wavelength converter. 1998 Elsevier Science B.V. All rights reserved. PACS: 34.39.th; 82.20.Rp; 33.50.!j Keywords: Fluorescence; Fluorescent probe; Wavelength converter; Artificial light-harvesting
1. Introduction Resonant excitation energy transfer (EET) between molecules as described by Fo¨rster theory [1] has been studied and applied from many perspectives [2]. The bimolecular rate of transfer depends on R\, where R is the distance between the donor and acceptor molecules, the overlap of the donor’s fluorescence and the acceptor’s absorption spectra and other factors. The most notable multi-dye EET
* Corresponding [email protected].
author.
Tel.:#1 814 898 6476;
e-mail:
networks are the light-harvesting pigment—protein systems of photosynthetic organisms [3]. These networks absorb sunlight predominately in the visible region and transfer their excitations to the reaction centers where photochemistry begins. The quantum efficiency of EET in these networks commonly exceeds 90% with collection over hundreds of pigments and single-step hopping times in the sub-picosecond region. Our interest is in determining whether or not large man-made multiple dye EET systems can be created with such high efficiencies. Many studies have observed EET between man-made configurations of dye molecules [2] in solutions [1,4,5],
0022-2313/97/$ — see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 2 3 1 3 ( 9 7 ) 0 0 0 4 4 - 1
226
D.V. Roberts et al. / Journal of Luminescence 79 (1998) 225—231
multi-layers [6] and solid environments [7], some with high efficiency. Langmuir—Blodgett multilayer films containing dyes have efficient transfer occurring between dyes separated by over 100 nm [6]. Efficient transfer between donor—acceptor systems of J-aggregates of dyes has also been observed [8—10]. These systems are typically composed of two dyes, one as the donor and the other as the acceptor. Chemically synthesized, supramolecular arrays of two or more linked dyes are the most successful of these artificial light-harvesting systems [11—16]. EET efficiencies of above 95% to the final fluorescing dye has been reported [15]. Applications for EET systems include their use in dye lasers [17,18], as probes of protein conformation and dynamics [4,19], as distance probes [4,20], as molecular wires [14] and optoelectronic gates [16]. Molecular Probes, Inc. has developed a series of fluorescent microspheres as fluorescent probes for use with biological systems [21]. The TransFluoSpheres威 [22] are polystyrene spheres available in a range of diameters that can be loaded with one or more types of dyes. They are designed to maximize EET among the dyes such that most of their emission comes from the lowest excited energy level dye in the spheres, independent of the wavelength of exciting light. Unlike the synthesized molecular arrays where dye molecules are bonded to each other [11—16], the spheres take a different approach by simply creating high enough concentrations of the dyes to facilitate efficient EET. The spheres offer optimal flexibility in choice of excitation and emission wavelengths and high fluorescence quantum yield. As such, they have been used as a fluorescent probe in a wide variety of experiments [21,23—25]. These spheres are wavelength converters, using EET to change absorbed light over a broad range into fluorescence at a narrow one. The spheres are a man-made light-harvesting network that work in a similar manner to those found in biological systems. Our goal is to determine just how efficient they are in this role through measurements of fluorescence, fluorescence excitation, and absorption. Our focus is on the 40 nm diameter spheres that contain six different types of dyes. With absorption over the entire visible region, they are most comparable to the breadth of absorption characteristic of photosynthetic light-harvesting EET networks.
2. Materials and methods The polystyrene microspheres used in this experiment were obtained from Molecular Probes Inc., 4849 Pitchford Ave. Eugene, OR 97402-9144 [21]. Our research centers on the study of the TransFluoSpheres威 Fluorescent Microspheres Model T-8869 [22]. Embedded within these 40 nm diameter spheres are six different derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY威) dyes whose absorption maxima vary from 505 to 688 nm [26—28]. Our letter designation to be used in this paper, the IUPAC names, molecular weights (MW), excitation and emission maxima (E /E ) and extinction coefficients (a) in solvent of these polyazaindacene dyes are as follows [26—28] (see Table 1). General structure and synthesis information on these [26—28] and similar dyes and how they are incorporated into the polystyrene spheres have been reported previously [21,22,29]. Fluorescence quantum yields for similar BODIPY derivatives in organic solvent range from 0.02 to nearly 1.0, with most being above 0.8 [22,29—31]. Their intrinsic radiative lifetimes are in the 5.3 to 6.5 ns region [29]. The six dyes (A through F) are randomly oriented and homogeneously dispersed within the spheres [22]. The relative concentrations of the dyes in the spheres have been adjusted empirically to maximize fluorescence from the dye with the longest wavelength emission (dye F) [22]. The relative concentrations of dyes in the Model T-8869 spheres are approximately 12 : 1.8 : 1.7 : 1.3: 1.0 : 1.1 for dyes A : B : C : D : E : F, respectively. To maximize excitation transfer and minimize non-radiative losses, the concentrations of the dyes in the spheres are such that the average intermolecular distance between any donor and acceptor dye molecule is between 2.5 to 4.0 nm [22]. Measurements from FluoSpheres威 Fluorescent Microspheres (Molecular Probes Inc., Models F-8823, F-8821, F-8820, F-8817, F-8816, F8807) [21] were used to provide absorption spectra for individual dyes in the spheres. These FluoSpheres威 are 1000 nm in diameter and contain only a single type of dye molecule. The microspheres were initially suspended in a buffer solution of distilled, de-ionized water with
D.V. Roberts et al. / Journal of Luminescence 79 (1998) 225—231
227
Table 1 Dye
Chemical Name [solvent]
MW (kD)
E /E (nm)
a (cm\ M\)
A
4,4-difluoro-1,3-dipropyl-4-bora-3a, 4a-diaza-s-indacene [ethanol] 4,4-difluoro-1,3-diphenyl-5,7-dipropyl -4-bora-3a,4a-diaza-sindacene [methanol] 4,4-difluoro-1,3,5,7-tetraphenyl-4-bora-3a, 4a-diaza-sindacene [methanol] 4,4-difluoro-1,3-diphenyl-5-(2-pyrrolyl) -4-bora-3a,4adiaza-s-indacene [methanol] difluoro(1-((3-(4-methyloxyphenyl)-2H-isoindol1-yl)methylene)-3-(4-methoxyphenyl) -1H-isoindolatoN,N)boron [methanol] difluoro(5-methoxy-1-((5-methoxy-3(4-methoxyphenyl)-2H-isoindol-1-yl)methylene)3-(4-methoxyphenyl)-1H-isoindolato-N,N)boron [chloroform]
276
490/515
65 600
428
540/560
76 400
498
564/592
78 000
409
600/635
81 700
504
645/678
95 500
564
675/720
113 500
B C D E
F
2 mM sodium azide and 0.02% Tween 20 at a pH of 7.5 at 4°C [21]. After being sonicated for 5 m, glycerol and additional buffer were added to bring samples to 80% glycerol by volume and an appropriate optical density for the measurement being taken. All measurements were taken with the samples at room temperature (23°C) in a quartz cuvette with a 1.5 mm path length. Fluorescence emission and fluorescence excitation spectra were measured using a Model QM-2 fluorimeter (Photon Technology International) with a Model R928 photomultiplier tube (Hamamatsu) in photon-counting mode. Emission was resolved with a bandwidth of 2 nm from 480 to 800 nm using an excitation wavelength of 470 nm with a 4 nm bandwidth for fluorescence measurements. The spectra were corrected for changes in the intensity of the excitation source during the measurement and for the wavelength response of the collection optics and detector. For fluorescence excitation measurements the excitation wavelength was scanned from 350 to 740 nm with a bandwidth of 2 nm. Fluorescence was collected at 750 nm with a bandwidth of 4 nm after passing through 520 and 730 nm long-pass filters, which function in blocking out any stray excitation light from entering the photomultiplier tube. The spectra were corrected for changes in the intensity of the excitation source caused by the passage of time and changing ex-
citation wavelength. To prevent scattering and re-absorption artifacts during fluorescence measurements the optical density of the solution was less than 0.12 at the absorption maximum of 505 nm. Absorption spectra were measured from 350 to 750 nm with a resolution of 2 nm using a Hewlett—Packard model 8452A diode array absorption spectrophotometer. To improve the signal-to-noise ratio the data were integrated over a time span of 24.5 s. Scans were taken twenty times on each sample to ensure reproducibility. The final absorption spectrum characterizing the six dyes had subtracted from it a reference absorption measurement that was taken on blank 40 nm polystyrene spheres containing no dyes. The concentration and solution were the same for both the blank and six-dye spheres. This subtraction was done to correct for the absorption of the polystyrene and some scattering in the ultraviolet part of the spectrum. For absorption measurements the sample’s optical density was 1.2 at 505 nm. The excitation transfer efficiency is the ratio of the number of excitations created in the lowest energy dye molecules (dye F) to the number of photons absorbed. This efficiency is calculated by dividing the fluorescence excitation spectrum for emission from the lowest energy dye alone by the absorption spectrum of the spheres.
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3. Results Fig. 1 shows the fluorescence spectrum of the six-dye spheres with excitation at 480 nm. A maximum is observed at 718 nm, which is the peak of emission from the lowest energy level dye. This large amount of fluorescence at this wavelength will be shown to be caused by highly efficient excitation transfer to the lowest energy dye. Small amounts of emission are observed between 480 and 650 nm with the next largest peak at 505 nm being 13 times smaller in amplitude than that at 718 nm. This indicates that the greater portion of the excitations are fluoresced by the lowest energy level dye. The absorption spectrum of the six-dye microspheres (Figs. 2 and 3) is characterized by five distinct peaks of different amplitudes. The first four peaks correspond to the first four dyes. The final peak, centered at 675 nm, is the combination of absorption by the final two dyes. Each polyazaindacene dye will be referred to by its designated letter. The dyes’ absorption and fluorescence emission maxima in the spheres (in that order) are given in parenthesis: dye A (505/516 nm), dye B (540/560 nm), dye C (577/607 nm), dye D (622/645 nm), dye E (659/680 nm), dye F (688/718 nm). The primary goal of this research is to determine the efficiency of excitation energy transfer from the five highest energy level dyes in the spheres (dyes A, B, C, D, and E) to the lowest energy dye molecules
Fig. 2. Absorption (- - - -) and fluorescence excitation spectra (—) of the six-dye TransFluoSpheres威 [22] (Molecular Probes Product T-8869). Fluorescence excitation spectrum was for emission from dye F at 750 nm. Also included is a plot of the ratio of the fluorescence excitation spectrum to the absorption spectrum (- - - -). When multiplied by 100%, it is a plot of the excitation transfer efficiency to dye F as a function of wavelength.
Fig. 3. Absorption spectrum of the six-dye TransFluoSpheres威 [22] (Molecular Probes Product T-8869) (—). Simplification of the six-dye TransFluoSpheres威 absorption spectra into absorption by individual dyes A (— -), B (— —), C (— ) — ) ), D (- - -), E (- - ) ) ), and F ( ) ) ) ) ). The inset is an expanded plot of the contributions of dyes C, D, E, and F to the long-wavelength end of the spectrum.
Fig. 1. The fluorescence spectrum of the six-dye TransFluoSpheres威 [22] (Molecular Probes Product T-8869). Sample is excited at 480 nm. Sample preparation is discussed in detail in the Materials and Methods section.
(dye F). To calculate this, the fluorescence excitation spectrum for emission from dye F alone is normalized to the absorption spectrum of the sixdye spheres at a location where only dye F directly absorbs light. The ratio of the two curves is then calculated to give the percent transfer. The logic here is that 100% of the photons that are absorbed
D.V. Roberts et al. / Journal of Luminescence 79 (1998) 225—231
by dye F create, by definition, excitations in the dye F molecules. Direct absorption is therefore equivalent to 100% excitation transfer. This gives us a place to normalize the curves since one now knows the resulting transfer efficiency in this range. Therefore, anywhere in the absorption spectrum where only dye F molecules absorb should, in theory, completely match the fluorescence excitation spectrum. A distinct absorption peak for dye F is not easily discernible from the absorption spectrum shown in Fig. 2. A region needed to be found where the absorption could be completely attributed to dye F and where normalization could be made. To find this region the contribution of each dye to the six-dye spheres’ total absorption spectrum was determined. An absorption spectrum for each dye was found by measuring the fluorescence excitation spectrum for a single dye contained in 1000 nm diameter spheres. Since these spheres contain only one dye, the absorption spectrum is the same as the fluorescence excitation spectrum for emission from that dye. Fig. 3 illustrates the deconvolution of the absorption spectrum of the six-dye spheres into contributions from each dye using the absorption spectra of the individual dyes. It shows that dye F accounts for over 99% of the absorption in the range of 706 to 750 nm making it possible to normalize anywhere in this range and get accurate results. Fig. 2 compares the fluorescence excitation spectrum for emission at 750 nm to the absorption spectrum of the six-dye spheres. In the range from 706 to 740 nm the two spectra line up completely within tolerance of the background noise. To minimize the effect of the background noise the normalization constant over this wavelength range was averaged. This is where absorption is dominated by dye F and, as discussed earlier, is equivalent to 100% EET. The ratio of the these two curves, which is shown as a line with small dashes, indicates the efficiency of excitation transfer. From 350 to 740 nm the curves highly overlap indicating high transfer efficiencies. As expected, the region from 706 to 740 nm is centered around 1 or 100% efficiency reflecting dominant absorption by dye F. The efficiency then drops to about 95% at 700 nm and remains flat at this level until 450 nm. The
229
excitation spectrum is slightly higher than the absorption spectrum just to the long wavelength side of the peak at 505 nm. It is also slightly lower than the absorption spectrum on the short wavelength side of the peak. This causes the excitation transfer efficiency curve to oscillate in this region and to go above 100%. This is an artifact caused by a slight mismatch of the locations of the peaks as measured by the two different instruments. The absorption spectrometer, with a resolution of 2 nm, limits our precision in this region of rapidly changing slope. The average excitation transfer efficiency was calculated to be 95% (standard deviation of 4%) from 450 to 740 nm and 86% (standard deviation 8%) from 350 to 450 nm.
4. Discussion In Fig. 2 the absorption and fluorescence excitation of the six dye spheres are seen to be nearly identical. This similarity is the result of highly efficient excitation transfer which averages out to be 95% efficient from 450 to 740 nm. Each individual dye by itself is highly fluorescent. When these dyes are put together in one sphere at the right concentrations they form a highly efficient excitation transfer network. The wide variety of spectral types results in the network having a wide total absorption spectrum covering the entire visible region. The absorption and fluorescence excitation spectra are identical in the range of 706 to 740 nm (Fig. 2). This confirms the hypothesis based on the results of Fig. 3 that only dye F is responsible for absorption beyond 706 nm and supports the assumption concerning where to normalize the two spectra. The high percentage of the fluorescence coming from dye F at 718 nm is a direct result of the large total transfer efficiency from dyes A through E to dye F (Fig. 1). A majority of the excitations in dyes A through E are transferred to dye F before they can result in emission from the former. Energy transfer dominates over radiative and other nonradiative processes for dyes A through E in this network. This also indicates that only a small fraction of the excitations created are lost to nonradiative quenching traps in the spheres, such as
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aggregates of the dyes. This occurs despite the fact that in order for such rapid excitation transfer to occur the dye molecules must be within approximately 4.0 nm of each other [22]. Resonant excitation energy transfer in these spheres likely follows Fo¨rster theory [1] based on conclusions from measurements of EET in dyedoped films of polystyrene [7]. In light of this, the dye molecules were chosen by Molecular Probes, Inc. to ensure good overlap of the donor molecule’s fluorescence with the acceptor molecule’s absorption for all six dyes. The relative concentrations of each dye in the spheres were also empirically adjusted to maximize the fluorescence from the lowest energy dye (dye F) [22]. The average spacing between donor and acceptor molecules of 2.5 to 4.0 nm [22] is consistent with Fo¨rster theory [1] and typical of distances needed to ensure high rates of excitation transfer [2]. Efficient EET systems are fundamental to photosynthetic organisms for the absorption and collection of solar energy [3]. These six-dye spheres rival pigmented photosynthetic antenna networks in terms of collection efficiency and absorption range. There are many similarities between the spheres and the light-harvesting extra-membrane particles of cyanobacteria called phycobilisomes [3,32,33]. Phycobilisomes contain many spectrally different types of dyes called phycobilins. The phycobilins are attached to aggregates of proteins that form sets of stacked disks. They create a multiple-dye EET network that absorbs light and efficiently funnels excitation energy down the stacks and into the chlorophylls of the photosystems that reside in thylakoid membrane. By using many different phycobilins, the phycobilisomes have a broad absorption covering the region of 450 to 650 nm where chlorophylls absorb weakly. The microspheres act as man-made phycobilisomes but do not have the complex and highly ordered structure of the phycobilisomes and their directional excitation transfer [3,32,33]. Though the spheres contain a random distribution of dyes, the lack of aggregates and the high concentration of molecules has created a situation for rapid Fo¨rster excitation transfer between the dyes such that virtually every excitation hops around the system until it reaches one of the lowest energy dye molecules.
The six-dye sphere EET network is unique among the man-made systems in consideration of the large number of different types of dyes used, its broad absorption spectrum and its high efficiency of EET. This system compliments the approach of modeling light-harvesting networks through the use of supramolecular dye arrays [11—16]. In the later, the composition and structural arrangement of the dyes can be defined and controlled, unlike in the spheres. The sphere format offers the chance to explore the kinetics of large ensembles of dyes as part of a light-harvesting network. Both defined arrangements of chromophores and large networks are properties of the natural light-harvesting systems that we need to understand further.
5. Conclusions Polystyrene spheres containing six types of dye form a highly efficient artificial light-harvesting network. The absorption spectrum of the spheres was nearly identical to the fluorescence excitation for emission from the lowest energy dye. This reflects the fact that 95% of photons absorbed over most of the visible region are transferred to the dye molecules with the lowest energy level through Fo¨rster resonant excitation transfer. Excitation anywhere within the spheres’ absorption spectrum results in fluorescence predominately at 718 nm from dye with the lowest energy level. The spheres are useful as a fluorescent probe in biological applications where flexibility in emission and excitation are required. They are also useful as artificial models for light-harvesting systems and wavelength converters in sensitization applications.
Acknowledgements Our appreciation to Molecular Probes, Inc. for providing samples for this research. We would like to thank many people from Molecular Probes who contributed their time and ideas in valuable discussions concerning our results. This work was supported by DVR from a research grant and by BPW from start-up funds through the Pennsylvania State University: Erie, The Behrend College.
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References [1] Th. Fo¨rster, in: O. Sinanoslu (Ed.), Modern Quantum Chemistry, Part III, Academic Press, New York, 1965, p. 93. [2] B.W. Van Der Meer, G. Coker, S.-Y. Chen, Resonance Energy Transfer: Theory and Data, VCH Publishers, Inc., New York, 1994. [3] R. van Grondelle, J.P. Dekker, T. Gillbro, V. Sundstrom, Biochem. Biophys. Acta 1187 (1994) 1. [4] J. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. [5] L.V. Levshin, B.T. Mukushev, A.M. Saletskii, Opt. Spect. 79 (1995) 564. [6] S. Draxler, M.E. Lippitsch, F.R. Aussenegg, Chem. Phys. Lett. 159 (1989) 231. [7] G.E. Johnson, Macromolecules 13 (1980) 145. [8] D. Mobius, Adv. Mater. 7 (1995) 437. [9] Y. Yonezawa, H. Kurokawa, T. Sato, J. Lumin. 54 (1993) 285. [10] M. Watanabe, M. Herren, M. Morita, J. Lumin. 58 (1994) 198. [11] J.S. Lindsey, P.A. Brown, D.A. Siesel, Tetrahedron 45 (1989) 4845. [12] J. Seth, V. Palaniappan, T.E. Johnson, S. Prathapan, J.S. Lindsey, D.F. Bocian, J. Am. Chem. Soc. 116 (1994) 10 578. [13] V.S.-Y. Lin, S.G. DiMagno, M.J. Therien, Highly Conjugated, Science 264 (1994) 1105. [14] R.W. Wagner, J.S. Lindsey, J. Am. Chem. Soc. 116 (1994) 9759. [15] J.-S. Hsiao, B.P. Krueger, R.W. Wagner, T.E. Johnson, J.K. Delaney, D.C. Mauzerall, G.R. Fleming, J.S. Lindsey, D.F. Bocian, R.J. Donohoe, J. Am. Chem. Soc. 118 (1996) 11 181.
231
[16] R.W. Wagner, J.S. Lindsey, J. Seth, V. Palaniappan, D.F. Bocian, J. Am. Chem. Soc. 118 (1996) 3996. [17] F.J. Duarte, L.W. Hillman, Dye Laser Principles, Academic Press, Boston, 1990. [18] D. Mohan, S. Sanghi, R.D. Singh, K. Mahendiratta, A. Guar, Photochem. Photobiol. A 68 (1992) 77. [19] B. Pispsia, V. Mariano, A. Pallwshchi, G. Zanotti, J. Molec. Liq. 61 (1994) 167. [20] M. Kaschke, B. Valeur, J. Bourson, N.P. Ernsting, Chem. Phys. Lett. 179 (1991) 544. [21] R.P. Haugland, Molecular Probes Product Information, CMP5001 1995. [22] J.M. Brinkley, R.P. Haugland, V.L. Singer, U.S. Patent No. 5326 692, 1994. [23] Y. Morita, L. Kaufman, K. Hosier, IEEE Trans. Nucl. Sci. 36 (1989) 978. [24] R. Glenny, S. Bernard, M. Brinkley, J. Appl. Physiol. 74 (1993) 2585. [25] H.T. Robertson, J. Appl. Physiol. 82 (1997) 943. [26] R.P. Haugland, H.C. Kang, U.S. Patent No. 4 774 339, 1988. [27] R.P. Haugland, H.C. Kang, U.S. Patent No. 5 248 782, 1993. [28] R.P. Haugland, in: B. Herman, K. Jacobson (Eds.), Optical Microscopy for Biology, Wiley-Liss, New York, 1990, p. 143. [29] J. Karolin, L.B.-A. Johansson, L. Strandberg, T. Ny, J. Am. Chem. Soc. 116 (1994) 7801. [30] I.A. Johnson, H.C. Kang, R.P. Haugland, Anal. Biochem. 198 (1991) 228. [31] R.W. Wagner, J.S. Lindsey, Pure Appl. Chem. 68 (1996) 1373. [32] E. Gantt, Annu. Rev. Plant Physiol. 32 (1981) 327. [33] R. MacColl, D. Guard-Friar, Phycobiliproteins, CRC Press, Boca Raton, FL, 1987.
Efficient excitation energy transfer among multiple dyes in polystyrene microspheres Daniel V. Roberts , Bruce P. Wittmershaus *, Yu-Zhong Zhang, Sharon Swan, Michael P. Klinosky School of Science, Pennsylvania State University: Erie, The Behrend College, Station Rd., Erie, PA 16563-0203, USA Molecular Probes Inc., 4849 Pitchford Ave. Eugene, OR 97402-9144, USA Received 27 December 1997; received in revised form 28 May 1998; accepted 10 July 1998
Abstract Our comparison of fluorescence excitation and absorption spectra from 40 nm polystyrene spheres containing six different dyes illustrates efficient excitation energy transfer among the dyes. The six-dye spheres collective absorption covers the entire visible region. The five dyes with the highest first excited state energy levels transfer their excitations to the sixth dye of the lowest energy level. The average excitation transfer efficiency to the sixth dye is 95% over most of the visible spectrum. As a result of this transfer, the spheres have a fluorescence maximum at 718 nm, the peak of emission from the lowest energy level dye. Each sphere functions as an artificial light-harvesting network. They have practical applications as fluorescent labels for biological systems and act as an efficient, broadly absorbing wavelength converter. 1998 Elsevier Science B.V. All rights reserved. PACS: 34.39.th; 82.20.Rp; 33.50.!j Keywords: Fluorescence; Fluorescent probe; Wavelength converter; Artificial light-harvesting
1. Introduction Resonant excitation energy transfer (EET) between molecules as described by Fo¨rster theory [1] has been studied and applied from many perspectives [2]. The bimolecular rate of transfer depends on R\, where R is the distance between the donor and acceptor molecules, the overlap of the donor’s fluorescence and the acceptor’s absorption spectra and other factors. The most notable multi-dye EET
* Corresponding [email protected].
author.
Tel.:#1 814 898 6476;
e-mail:
networks are the light-harvesting pigment—protein systems of photosynthetic organisms [3]. These networks absorb sunlight predominately in the visible region and transfer their excitations to the reaction centers where photochemistry begins. The quantum efficiency of EET in these networks commonly exceeds 90% with collection over hundreds of pigments and single-step hopping times in the sub-picosecond region. Our interest is in determining whether or not large man-made multiple dye EET systems can be created with such high efficiencies. Many studies have observed EET between man-made configurations of dye molecules [2] in solutions [1,4,5],
0022-2313/97/$ — see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 2 3 1 3 ( 9 7 ) 0 0 0 4 4 - 1
226
D.V. Roberts et al. / Journal of Luminescence 79 (1998) 225—231
multi-layers [6] and solid environments [7], some with high efficiency. Langmuir—Blodgett multilayer films containing dyes have efficient transfer occurring between dyes separated by over 100 nm [6]. Efficient transfer between donor—acceptor systems of J-aggregates of dyes has also been observed [8—10]. These systems are typically composed of two dyes, one as the donor and the other as the acceptor. Chemically synthesized, supramolecular arrays of two or more linked dyes are the most successful of these artificial light-harvesting systems [11—16]. EET efficiencies of above 95% to the final fluorescing dye has been reported [15]. Applications for EET systems include their use in dye lasers [17,18], as probes of protein conformation and dynamics [4,19], as distance probes [4,20], as molecular wires [14] and optoelectronic gates [16]. Molecular Probes, Inc. has developed a series of fluorescent microspheres as fluorescent probes for use with biological systems [21]. The TransFluoSpheres威 [22] are polystyrene spheres available in a range of diameters that can be loaded with one or more types of dyes. They are designed to maximize EET among the dyes such that most of their emission comes from the lowest excited energy level dye in the spheres, independent of the wavelength of exciting light. Unlike the synthesized molecular arrays where dye molecules are bonded to each other [11—16], the spheres take a different approach by simply creating high enough concentrations of the dyes to facilitate efficient EET. The spheres offer optimal flexibility in choice of excitation and emission wavelengths and high fluorescence quantum yield. As such, they have been used as a fluorescent probe in a wide variety of experiments [21,23—25]. These spheres are wavelength converters, using EET to change absorbed light over a broad range into fluorescence at a narrow one. The spheres are a man-made light-harvesting network that work in a similar manner to those found in biological systems. Our goal is to determine just how efficient they are in this role through measurements of fluorescence, fluorescence excitation, and absorption. Our focus is on the 40 nm diameter spheres that contain six different types of dyes. With absorption over the entire visible region, they are most comparable to the breadth of absorption characteristic of photosynthetic light-harvesting EET networks.
2. Materials and methods The polystyrene microspheres used in this experiment were obtained from Molecular Probes Inc., 4849 Pitchford Ave. Eugene, OR 97402-9144 [21]. Our research centers on the study of the TransFluoSpheres威 Fluorescent Microspheres Model T-8869 [22]. Embedded within these 40 nm diameter spheres are six different derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY威) dyes whose absorption maxima vary from 505 to 688 nm [26—28]. Our letter designation to be used in this paper, the IUPAC names, molecular weights (MW), excitation and emission maxima (E /E ) and extinction coefficients (a) in solvent of these polyazaindacene dyes are as follows [26—28] (see Table 1). General structure and synthesis information on these [26—28] and similar dyes and how they are incorporated into the polystyrene spheres have been reported previously [21,22,29]. Fluorescence quantum yields for similar BODIPY derivatives in organic solvent range from 0.02 to nearly 1.0, with most being above 0.8 [22,29—31]. Their intrinsic radiative lifetimes are in the 5.3 to 6.5 ns region [29]. The six dyes (A through F) are randomly oriented and homogeneously dispersed within the spheres [22]. The relative concentrations of the dyes in the spheres have been adjusted empirically to maximize fluorescence from the dye with the longest wavelength emission (dye F) [22]. The relative concentrations of dyes in the Model T-8869 spheres are approximately 12 : 1.8 : 1.7 : 1.3: 1.0 : 1.1 for dyes A : B : C : D : E : F, respectively. To maximize excitation transfer and minimize non-radiative losses, the concentrations of the dyes in the spheres are such that the average intermolecular distance between any donor and acceptor dye molecule is between 2.5 to 4.0 nm [22]. Measurements from FluoSpheres威 Fluorescent Microspheres (Molecular Probes Inc., Models F-8823, F-8821, F-8820, F-8817, F-8816, F8807) [21] were used to provide absorption spectra for individual dyes in the spheres. These FluoSpheres威 are 1000 nm in diameter and contain only a single type of dye molecule. The microspheres were initially suspended in a buffer solution of distilled, de-ionized water with
D.V. Roberts et al. / Journal of Luminescence 79 (1998) 225—231
227
Table 1 Dye
Chemical Name [solvent]
MW (kD)
E /E (nm)
a (cm\ M\)
A
4,4-difluoro-1,3-dipropyl-4-bora-3a, 4a-diaza-s-indacene [ethanol] 4,4-difluoro-1,3-diphenyl-5,7-dipropyl -4-bora-3a,4a-diaza-sindacene [methanol] 4,4-difluoro-1,3,5,7-tetraphenyl-4-bora-3a, 4a-diaza-sindacene [methanol] 4,4-difluoro-1,3-diphenyl-5-(2-pyrrolyl) -4-bora-3a,4adiaza-s-indacene [methanol] difluoro(1-((3-(4-methyloxyphenyl)-2H-isoindol1-yl)methylene)-3-(4-methoxyphenyl) -1H-isoindolatoN,N)boron [methanol] difluoro(5-methoxy-1-((5-methoxy-3(4-methoxyphenyl)-2H-isoindol-1-yl)methylene)3-(4-methoxyphenyl)-1H-isoindolato-N,N)boron [chloroform]
276
490/515
65 600
428
540/560
76 400
498
564/592
78 000
409
600/635
81 700
504
645/678
95 500
564
675/720
113 500
B C D E
F
2 mM sodium azide and 0.02% Tween 20 at a pH of 7.5 at 4°C [21]. After being sonicated for 5 m, glycerol and additional buffer were added to bring samples to 80% glycerol by volume and an appropriate optical density for the measurement being taken. All measurements were taken with the samples at room temperature (23°C) in a quartz cuvette with a 1.5 mm path length. Fluorescence emission and fluorescence excitation spectra were measured using a Model QM-2 fluorimeter (Photon Technology International) with a Model R928 photomultiplier tube (Hamamatsu) in photon-counting mode. Emission was resolved with a bandwidth of 2 nm from 480 to 800 nm using an excitation wavelength of 470 nm with a 4 nm bandwidth for fluorescence measurements. The spectra were corrected for changes in the intensity of the excitation source during the measurement and for the wavelength response of the collection optics and detector. For fluorescence excitation measurements the excitation wavelength was scanned from 350 to 740 nm with a bandwidth of 2 nm. Fluorescence was collected at 750 nm with a bandwidth of 4 nm after passing through 520 and 730 nm long-pass filters, which function in blocking out any stray excitation light from entering the photomultiplier tube. The spectra were corrected for changes in the intensity of the excitation source caused by the passage of time and changing ex-
citation wavelength. To prevent scattering and re-absorption artifacts during fluorescence measurements the optical density of the solution was less than 0.12 at the absorption maximum of 505 nm. Absorption spectra were measured from 350 to 750 nm with a resolution of 2 nm using a Hewlett—Packard model 8452A diode array absorption spectrophotometer. To improve the signal-to-noise ratio the data were integrated over a time span of 24.5 s. Scans were taken twenty times on each sample to ensure reproducibility. The final absorption spectrum characterizing the six dyes had subtracted from it a reference absorption measurement that was taken on blank 40 nm polystyrene spheres containing no dyes. The concentration and solution were the same for both the blank and six-dye spheres. This subtraction was done to correct for the absorption of the polystyrene and some scattering in the ultraviolet part of the spectrum. For absorption measurements the sample’s optical density was 1.2 at 505 nm. The excitation transfer efficiency is the ratio of the number of excitations created in the lowest energy dye molecules (dye F) to the number of photons absorbed. This efficiency is calculated by dividing the fluorescence excitation spectrum for emission from the lowest energy dye alone by the absorption spectrum of the spheres.
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3. Results Fig. 1 shows the fluorescence spectrum of the six-dye spheres with excitation at 480 nm. A maximum is observed at 718 nm, which is the peak of emission from the lowest energy level dye. This large amount of fluorescence at this wavelength will be shown to be caused by highly efficient excitation transfer to the lowest energy dye. Small amounts of emission are observed between 480 and 650 nm with the next largest peak at 505 nm being 13 times smaller in amplitude than that at 718 nm. This indicates that the greater portion of the excitations are fluoresced by the lowest energy level dye. The absorption spectrum of the six-dye microspheres (Figs. 2 and 3) is characterized by five distinct peaks of different amplitudes. The first four peaks correspond to the first four dyes. The final peak, centered at 675 nm, is the combination of absorption by the final two dyes. Each polyazaindacene dye will be referred to by its designated letter. The dyes’ absorption and fluorescence emission maxima in the spheres (in that order) are given in parenthesis: dye A (505/516 nm), dye B (540/560 nm), dye C (577/607 nm), dye D (622/645 nm), dye E (659/680 nm), dye F (688/718 nm). The primary goal of this research is to determine the efficiency of excitation energy transfer from the five highest energy level dyes in the spheres (dyes A, B, C, D, and E) to the lowest energy dye molecules
Fig. 2. Absorption (- - - -) and fluorescence excitation spectra (—) of the six-dye TransFluoSpheres威 [22] (Molecular Probes Product T-8869). Fluorescence excitation spectrum was for emission from dye F at 750 nm. Also included is a plot of the ratio of the fluorescence excitation spectrum to the absorption spectrum (- - - -). When multiplied by 100%, it is a plot of the excitation transfer efficiency to dye F as a function of wavelength.
Fig. 3. Absorption spectrum of the six-dye TransFluoSpheres威 [22] (Molecular Probes Product T-8869) (—). Simplification of the six-dye TransFluoSpheres威 absorption spectra into absorption by individual dyes A (— -), B (— —), C (— ) — ) ), D (- - -), E (- - ) ) ), and F ( ) ) ) ) ). The inset is an expanded plot of the contributions of dyes C, D, E, and F to the long-wavelength end of the spectrum.
Fig. 1. The fluorescence spectrum of the six-dye TransFluoSpheres威 [22] (Molecular Probes Product T-8869). Sample is excited at 480 nm. Sample preparation is discussed in detail in the Materials and Methods section.
(dye F). To calculate this, the fluorescence excitation spectrum for emission from dye F alone is normalized to the absorption spectrum of the sixdye spheres at a location where only dye F directly absorbs light. The ratio of the two curves is then calculated to give the percent transfer. The logic here is that 100% of the photons that are absorbed
D.V. Roberts et al. / Journal of Luminescence 79 (1998) 225—231
by dye F create, by definition, excitations in the dye F molecules. Direct absorption is therefore equivalent to 100% excitation transfer. This gives us a place to normalize the curves since one now knows the resulting transfer efficiency in this range. Therefore, anywhere in the absorption spectrum where only dye F molecules absorb should, in theory, completely match the fluorescence excitation spectrum. A distinct absorption peak for dye F is not easily discernible from the absorption spectrum shown in Fig. 2. A region needed to be found where the absorption could be completely attributed to dye F and where normalization could be made. To find this region the contribution of each dye to the six-dye spheres’ total absorption spectrum was determined. An absorption spectrum for each dye was found by measuring the fluorescence excitation spectrum for a single dye contained in 1000 nm diameter spheres. Since these spheres contain only one dye, the absorption spectrum is the same as the fluorescence excitation spectrum for emission from that dye. Fig. 3 illustrates the deconvolution of the absorption spectrum of the six-dye spheres into contributions from each dye using the absorption spectra of the individual dyes. It shows that dye F accounts for over 99% of the absorption in the range of 706 to 750 nm making it possible to normalize anywhere in this range and get accurate results. Fig. 2 compares the fluorescence excitation spectrum for emission at 750 nm to the absorption spectrum of the six-dye spheres. In the range from 706 to 740 nm the two spectra line up completely within tolerance of the background noise. To minimize the effect of the background noise the normalization constant over this wavelength range was averaged. This is where absorption is dominated by dye F and, as discussed earlier, is equivalent to 100% EET. The ratio of the these two curves, which is shown as a line with small dashes, indicates the efficiency of excitation transfer. From 350 to 740 nm the curves highly overlap indicating high transfer efficiencies. As expected, the region from 706 to 740 nm is centered around 1 or 100% efficiency reflecting dominant absorption by dye F. The efficiency then drops to about 95% at 700 nm and remains flat at this level until 450 nm. The
229
excitation spectrum is slightly higher than the absorption spectrum just to the long wavelength side of the peak at 505 nm. It is also slightly lower than the absorption spectrum on the short wavelength side of the peak. This causes the excitation transfer efficiency curve to oscillate in this region and to go above 100%. This is an artifact caused by a slight mismatch of the locations of the peaks as measured by the two different instruments. The absorption spectrometer, with a resolution of 2 nm, limits our precision in this region of rapidly changing slope. The average excitation transfer efficiency was calculated to be 95% (standard deviation of 4%) from 450 to 740 nm and 86% (standard deviation 8%) from 350 to 450 nm.
4. Discussion In Fig. 2 the absorption and fluorescence excitation of the six dye spheres are seen to be nearly identical. This similarity is the result of highly efficient excitation transfer which averages out to be 95% efficient from 450 to 740 nm. Each individual dye by itself is highly fluorescent. When these dyes are put together in one sphere at the right concentrations they form a highly efficient excitation transfer network. The wide variety of spectral types results in the network having a wide total absorption spectrum covering the entire visible region. The absorption and fluorescence excitation spectra are identical in the range of 706 to 740 nm (Fig. 2). This confirms the hypothesis based on the results of Fig. 3 that only dye F is responsible for absorption beyond 706 nm and supports the assumption concerning where to normalize the two spectra. The high percentage of the fluorescence coming from dye F at 718 nm is a direct result of the large total transfer efficiency from dyes A through E to dye F (Fig. 1). A majority of the excitations in dyes A through E are transferred to dye F before they can result in emission from the former. Energy transfer dominates over radiative and other nonradiative processes for dyes A through E in this network. This also indicates that only a small fraction of the excitations created are lost to nonradiative quenching traps in the spheres, such as
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aggregates of the dyes. This occurs despite the fact that in order for such rapid excitation transfer to occur the dye molecules must be within approximately 4.0 nm of each other [22]. Resonant excitation energy transfer in these spheres likely follows Fo¨rster theory [1] based on conclusions from measurements of EET in dyedoped films of polystyrene [7]. In light of this, the dye molecules were chosen by Molecular Probes, Inc. to ensure good overlap of the donor molecule’s fluorescence with the acceptor molecule’s absorption for all six dyes. The relative concentrations of each dye in the spheres were also empirically adjusted to maximize the fluorescence from the lowest energy dye (dye F) [22]. The average spacing between donor and acceptor molecules of 2.5 to 4.0 nm [22] is consistent with Fo¨rster theory [1] and typical of distances needed to ensure high rates of excitation transfer [2]. Efficient EET systems are fundamental to photosynthetic organisms for the absorption and collection of solar energy [3]. These six-dye spheres rival pigmented photosynthetic antenna networks in terms of collection efficiency and absorption range. There are many similarities between the spheres and the light-harvesting extra-membrane particles of cyanobacteria called phycobilisomes [3,32,33]. Phycobilisomes contain many spectrally different types of dyes called phycobilins. The phycobilins are attached to aggregates of proteins that form sets of stacked disks. They create a multiple-dye EET network that absorbs light and efficiently funnels excitation energy down the stacks and into the chlorophylls of the photosystems that reside in thylakoid membrane. By using many different phycobilins, the phycobilisomes have a broad absorption covering the region of 450 to 650 nm where chlorophylls absorb weakly. The microspheres act as man-made phycobilisomes but do not have the complex and highly ordered structure of the phycobilisomes and their directional excitation transfer [3,32,33]. Though the spheres contain a random distribution of dyes, the lack of aggregates and the high concentration of molecules has created a situation for rapid Fo¨rster excitation transfer between the dyes such that virtually every excitation hops around the system until it reaches one of the lowest energy dye molecules.
The six-dye sphere EET network is unique among the man-made systems in consideration of the large number of different types of dyes used, its broad absorption spectrum and its high efficiency of EET. This system compliments the approach of modeling light-harvesting networks through the use of supramolecular dye arrays [11—16]. In the later, the composition and structural arrangement of the dyes can be defined and controlled, unlike in the spheres. The sphere format offers the chance to explore the kinetics of large ensembles of dyes as part of a light-harvesting network. Both defined arrangements of chromophores and large networks are properties of the natural light-harvesting systems that we need to understand further.
5. Conclusions Polystyrene spheres containing six types of dye form a highly efficient artificial light-harvesting network. The absorption spectrum of the spheres was nearly identical to the fluorescence excitation for emission from the lowest energy dye. This reflects the fact that 95% of photons absorbed over most of the visible region are transferred to the dye molecules with the lowest energy level through Fo¨rster resonant excitation transfer. Excitation anywhere within the spheres’ absorption spectrum results in fluorescence predominately at 718 nm from dye with the lowest energy level. The spheres are useful as a fluorescent probe in biological applications where flexibility in emission and excitation are required. They are also useful as artificial models for light-harvesting systems and wavelength converters in sensitization applications.
Acknowledgements Our appreciation to Molecular Probes, Inc. for providing samples for this research. We would like to thank many people from Molecular Probes who contributed their time and ideas in valuable discussions concerning our results. This work was supported by DVR from a research grant and by BPW from start-up funds through the Pennsylvania State University: Erie, The Behrend College.
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References [1] Th. Fo¨rster, in: O. Sinanoslu (Ed.), Modern Quantum Chemistry, Part III, Academic Press, New York, 1965, p. 93. [2] B.W. Van Der Meer, G. Coker, S.-Y. Chen, Resonance Energy Transfer: Theory and Data, VCH Publishers, Inc., New York, 1994. [3] R. van Grondelle, J.P. Dekker, T. Gillbro, V. Sundstrom, Biochem. Biophys. Acta 1187 (1994) 1. [4] J. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. [5] L.V. Levshin, B.T. Mukushev, A.M. Saletskii, Opt. Spect. 79 (1995) 564. [6] S. Draxler, M.E. Lippitsch, F.R. Aussenegg, Chem. Phys. Lett. 159 (1989) 231. [7] G.E. Johnson, Macromolecules 13 (1980) 145. [8] D. Mobius, Adv. Mater. 7 (1995) 437. [9] Y. Yonezawa, H. Kurokawa, T. Sato, J. Lumin. 54 (1993) 285. [10] M. Watanabe, M. Herren, M. Morita, J. Lumin. 58 (1994) 198. [11] J.S. Lindsey, P.A. Brown, D.A. Siesel, Tetrahedron 45 (1989) 4845. [12] J. Seth, V. Palaniappan, T.E. Johnson, S. Prathapan, J.S. Lindsey, D.F. Bocian, J. Am. Chem. Soc. 116 (1994) 10 578. [13] V.S.-Y. Lin, S.G. DiMagno, M.J. Therien, Highly Conjugated, Science 264 (1994) 1105. [14] R.W. Wagner, J.S. Lindsey, J. Am. Chem. Soc. 116 (1994) 9759. [15] J.-S. Hsiao, B.P. Krueger, R.W. Wagner, T.E. Johnson, J.K. Delaney, D.C. Mauzerall, G.R. Fleming, J.S. Lindsey, D.F. Bocian, R.J. Donohoe, J. Am. Chem. Soc. 118 (1996) 11 181.
231
[16] R.W. Wagner, J.S. Lindsey, J. Seth, V. Palaniappan, D.F. Bocian, J. Am. Chem. Soc. 118 (1996) 3996. [17] F.J. Duarte, L.W. Hillman, Dye Laser Principles, Academic Press, Boston, 1990. [18] D. Mohan, S. Sanghi, R.D. Singh, K. Mahendiratta, A. Guar, Photochem. Photobiol. A 68 (1992) 77. [19] B. Pispsia, V. Mariano, A. Pallwshchi, G. Zanotti, J. Molec. Liq. 61 (1994) 167. [20] M. Kaschke, B. Valeur, J. Bourson, N.P. Ernsting, Chem. Phys. Lett. 179 (1991) 544. [21] R.P. Haugland, Molecular Probes Product Information, CMP5001 1995. [22] J.M. Brinkley, R.P. Haugland, V.L. Singer, U.S. Patent No. 5326 692, 1994. [23] Y. Morita, L. Kaufman, K. Hosier, IEEE Trans. Nucl. Sci. 36 (1989) 978. [24] R. Glenny, S. Bernard, M. Brinkley, J. Appl. Physiol. 74 (1993) 2585. [25] H.T. Robertson, J. Appl. Physiol. 82 (1997) 943. [26] R.P. Haugland, H.C. Kang, U.S. Patent No. 4 774 339, 1988. [27] R.P. Haugland, H.C. Kang, U.S. Patent No. 5 248 782, 1993. [28] R.P. Haugland, in: B. Herman, K. Jacobson (Eds.), Optical Microscopy for Biology, Wiley-Liss, New York, 1990, p. 143. [29] J. Karolin, L.B.-A. Johansson, L. Strandberg, T. Ny, J. Am. Chem. Soc. 116 (1994) 7801. [30] I.A. Johnson, H.C. Kang, R.P. Haugland, Anal. Biochem. 198 (1991) 228. [31] R.W. Wagner, J.S. Lindsey, Pure Appl. Chem. 68 (1996) 1373. [32] E. Gantt, Annu. Rev. Plant Physiol. 32 (1981) 327. [33] R. MacColl, D. Guard-Friar, Phycobiliproteins, CRC Press, Boca Raton, FL, 1987.