[August 12, 2011] Quantum Yields and Quantitative Spectra of Firefly Bioluminescence with Various Bivalent Metal Ions [Photochemistry and Photobiology] Tweet (Photochemistry and Photobiology Via Acquire Media NewsEdge) ABSTRACT We measured quantitative spectra of firefly (Photinus pyralis) bioluminescence in the presence of Zn2+ and other bivalent metal ions to investigate the effects of these metal ions on luciferin-luciferase reaction. We studied the dependence of the quantum yield and spectrum on quantity and kind of bivalent metal ions. Adding various amounts of Mg2+, Mn2+ and Ca2+ produced virtually no change in the quantum yields or the spectra of bioluminescence. In contrast, increasing amounts of ions such as Zn2+ and Cd2+ decreased quantum yields and changed the bioluminescence color from yellow-green to red. Quantitative analysis showed that the sensitivities of the quantum yield and color to various metal ions were in the order of Hg2+ >Zn2+, Cd2+ > Ni2+, Co2+, Fe2+ [much greater than] Mg2+, Mn2+, Ca2+. We propose that the changes in quantum yield and spectrum caused by the metal ions are due to their effect on luciferase that surrounds oxyluciferin during its radioactive decay. We also found that having more metal ions accelerated bioluminescence reactions. The sensitivity of the reaction rate had no correlation with those of the quantum yield and spectrum. INTRODUCTION Quantitative studies of the fundamental properties of firefly bioluminescence have attracted increasing interest among researchers in various fields. Basic science still faces long-standing issues in describing the mechanisms behind the color change and high quantum yield of firefly bioluminescence; quantitatively measuring bioluminescence spectra, instead of the relative or normalized bioluminescence spectra, would be useful for detailed studies, particularly comparisons with modern quantum-chemistry calculations. In various bioluminescence applications, such as bioluminescence imaging, gene reporting and environmental monitoring (1), bioluminescence intensities are used to image or quantify the amount of target materials. However, bioluminescence intensities are affected not only by the amount of the target materials but also by the activity of the bioluminescence assay used in the analysis and the activity of the bioluminescence assay is in turn determined by the product of the quantum yield and reaction rate. Thus, quantitative analysis and knowledge of the quantum yield and reaction rate under various reaction conditions are very important for advanced applications of bioluminescence and for the development of assays. Bivalent metal ions are a cofactor in enzymatic reactions of firefly bioluminescence. The most commonly used bivalent metal ion is Mg2+, although some other ions have also been examined to activate or inhibit the reaction (2-4). We should note, however, that the effects of those different ions have been evaluated only in terms of the initial activities of the enzymatic reaction of bioluminescence, in other words, by measuring the bioluminescence intensity just after starting the reaction by mixing all solutions. Separate and quantitative evaluations of the quantum yield and reaction rate have never been conducted. Since various bivalent metal ions including Mg2+ often coexist inside living cells, quantitative evaluation of the influence of these metal ions is indispensable for intracellular applications of firefly bioluminescence. Bivalent metal ions are known to assist the luciferin-ATP activation reaction to form an intermediate (luciferyl-AMP). However, other possible effects on luciferase, and hence, on the bioluminescence spectra and quantum yields have not been well studied. In addition, some bivalent metal ions, such as Zn2+ and Cd2+, are known to shift the bioluminescence spectral peak to red (2). Hence, a systematic study of effects of these ions may provide a clue to the mechanism of the color changes caused by pH (5) and by residue-substituted luciferase (6-8). Ando et al. developed a spectrometer system that measures the quantitative total-photon-flux bioluminescence spectra (9) and quantitatively measured and analyzed the pH-dependent properties of the bioluminescence spectra of the North American firefly (Photinus pyralis) (10). They found the quantum yield in firefly bioluminescence to be 41.0% ± 7.4%, in contrast to the prevailing value of 88% ± 25% or nearly 100% (5). They also found that all the measured quantitative bioluminescence spectra could be systematically decomposed into one pH-sensitive and two pH-insensitive Gaussian components, and that merely the intensity variation of the pH-sensitive Gaussian component peaking at 2.2 eV, instead of the intensity conversion between yellow-green and red emissions via pH equilibrium, caused the changes in the apparent emission colors. In this study, we quantitatively investigated the influence of bivalent metal cations separately on the quantum yield, spectrum and reaction rate of the North American firefly (P. pyralis) bioluminescence reaction. We revised the setup from the previous study (9), made a new and independent calibration, and measured the quantum yields, quantitative spectra and reaction rates in the presence of various amounts and kinds of bivalent metal ions. Then we analyzed the spectra by means of curve fitting and clarified the sensitivity differences in the firefly bioluminescence spectra to bivalent metal ions. MATERIALS AND METHODS Chemicals and in vitro bioluminescence reaction. D-luciferin sodium salt (HPLC 99.0% purity; Wako) was diluted to 7.5 × 10-8 M with GTA-NaOH buffer (consisting of 0.05 M 3,3-dimethylglutaric acid, 0.05 M 2-amino-2-hydroxymethyl-1,3-protanediol and 0.05 M 2-amino-2-methyl-1,3-propanediol, adjusted to pH 8.0 with NaOH) within 4 h before measurement. The accurate concentration was characterized by absorption measurements using the log of the optical density log (OD^sub 327 nm^) = 4.27(11). The luciferase solution of the estimated concentration of 2 × 10-5 M was prepared by dissolving 1 mg of firefly (P. pyralis) luciferase powder (Sigma, molecular weight of about 60 kDa, purified from natural firefly) with 1 mL of 0.15 M GTA buffer containing 10% glycerol. ATP (adenosine-5'-triphospate disodium salt trihydrate, 99% purity; MP Biomedicals) aqueous solution was adjusted to pH 8.0 with NaOH and then diluted to 1 × 10-3 M with GTA buffer of pH 8.0. Bivalent metal salts (MgSO4, CaCl2, MnCl2, FeCl2, Co(NO3)2, NiCl2, ZnSO4, CdCl2 and HgCl2 [Wako]) were all reagent grade and were diluted to the required concentration with ultrapure water. The concentrations and volumes of reagents used for bioluminescence measurement are listed in Table 1. Note that we chose the condition that the final concentration of luciferin, 3.8 × 10-9 M is much lower than that of luciferase, 1 × 10-6 M to ensure that the concentration of available luciferase slays approximately constant during the reaction process. To start and hold the reaction for the bioluminescence measurements, a luciferin-luciferase solution was prepared by mixing all the solutions except for ATP, and the ATP solution was added to it as a trigger solution. Alternatively, the metal salt solution was premixed with ATP in the trigger solution, instead of in the luciferin-luciferase solution, to avoid probable gradual inactivation of luciferase due to its combination with metal ions. The pH values of all solutions were reexamined after the reaction; all fell in the range of 8.00 ± 0.08 (SD). The same samples were simultaneously measured with a luminometer. After the bioluminescence measurement, we added extra luciferase to the solution and estimated the residual luciferin by measuring the remaining bioluminescence intensity. In this manner, we confirmed that over 99% of the D-luciferin was consumed. Reaction rate measurement. We measured the time traces of the bioluminescence intensity with a luminometer (ATTO, AB-2200) and obtained the decay time of bioluminescence intensity from single exponential decay curves. Quantitative bioluminescence measurement setup. The quantitative bioluminescence spectra were obtained with a calibrated total-photon-flux charge coupled device (CCD)-spectrometer system as was done in reference (9). We used a homemade sample cell, the light-collection efficiency of which could be obtained by comparing stable luminescent light intensity in it and in a double-plate cell; the light-collection efficiency of the latter was geometrically calculated. The light given out by the bioluminescent solution was dispersed by a spectrometer (SpectraPro 300i; Acton Research) and collected by a back-illuminated CCD detector (Spec-10:400BR; Princeton Instruments) with higher full-well capacity than the previous one. The CCD was cooled with liquid nitrogen and operated at low temperature (-120°C). We calibrated the light-collection efficiency of the sample cell, the transmissivity of the slit in the spectrometer, and the absolute spectroscopy sensitivity of this setup in the same way as in reference (9). We replaced the power meter with a photodiode (calibrated by AIST, Japan) in the spectroscopy sensitivity calibration, and this reduced the uncertainty of the power measurement from 5% to 0.9%. Calibration and quantum-yield estimation. The light-collection efficiency of the acrylate tube cell for the bioluminescence measurement was 0.279% ± 0.015% (SD) and the transmissivity of the slit was 5.74% ± 0.08% in our measurement system. The absolute sensitivity of the system was 19.6 ± 1.7 photons per count, and this value takes account of the transmissivity of the lens and mirrors. We calculated the absolute number of photons emitted from 2.26 × 1011 luciferin molecules from the time-integrated spectra and derived the quantum yields. With the above calibrations, we calculated the quantum yield of firefly bioluminescence to be 47.6% ± 2.2% (SD) in the presence of 5 mM of Mg2+ at pH 8.0. The statistical deviation is much less than the 15% uncertainty of the calibration, which is 7.2% for the 47.6% value. Thus, we concluded the quantum yield to be 47.6% ± 7.2% (coverage factor k = 1). This value is thus within the estimated uncertainty of the previous reported value of 41.0% ± 7.4% by Y. Ando et al. (10). All the measurements were conducted at 24 ± 1°C. RESULTS Quantum yield All the bivalent metal ions that we investigated (Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+ and Hg2+) can be used instead of Mg2+ to catalyze firefly luciferin-luciferase reaction and to generate bioluminescence. In the measurements with Mg2+, Ca2+ and Mn2+, premixing of the metal salt solutions with the luciferin-luciferase solutions or with the ATP trigger solutions did not cause any differences in the experimental results. However, the results were different between the two cases in the measurements with Fe2+, Co2+, Ni2+, Zn2+, Cd2+ and Hg2+: preincubation of these metal salt solutions with the luciferin-luciferase solution slightly decreased quantum yields and greatly decelerated the reaction. The data shown below for these metal ions were obtained by premixing them into the ATP trigger solutions to avoid gradual inactivation of luciferase. Figure 1 shows the plots of bioluminescence quantum yield against the concentrations of various metal ions. It demonstrates that quantum yield depends on the amount and kind of metal ions. Quantum yield was not sensitive to the concentrations of Mg2+, Ca2+ and Mn2+, as shown in panel (a) of Fig. 1: Quantum yield increased only slightly with higher Mg2+ concentrations, and decreased very slightly with higher Mn2+ and Ca2+ concentrations. In contrast, quantum yield decreased steeply with higher concentrations of Zn2+ and other transition metal ions (i.e. Fe2+, Ni2+ , Co2+, Cd2+ and Hg2+), as shown in panel (b) of Fig. 1. It dropped to less than 10% in the presence of 3 mM of Zn2+ and Cd2+; but it remained at a relatively high value (over 20%) when equal amounts of Ni2+, Co2+ and Fe2+ ions were included in the reaction. Moreover, it had already dropped below 5% when 2.5 × 10-5 M of Hg2+ ions was present. Quantum yield was much more sensitive to the amount of Hg2+ than to other metal ions. Spectrum Similar to the quantum yield, the shape and peak of spectra changed very slightly as a result of increasing the amounts of Mg2+, Ca2+ and Mn2+ ions. On the other hand, we observed that spectral peak shifted from 560 nm to 620 nm by adding proper amounts of Zn2+, Cd2+, Fe2+, Co2+, Ni2+ and Hg2+, which had caused the quantum yield to decrease. The spectra in the presence of various amounts of Zn2+ are shown in Fig. 2. The spectrum of the reaction involving 5 mM of Mg2+ is also included in the plot as a reference of intensity and spectral shape. Increasing the Zn2+ amount from 0.1 to 1 mM resulted in a gradual spectral-peak shift from 560 to 620 nm. Quantitative analysis on spectra We performed Gaussian curve fitting to analyze the color dependence on the amount of metal ions. As shown in Fig. 3, all the bioluminescence spectra are well reproduced by assuming three Gaussian components, in which changes in the peak energy and full width at half maximum are negligibly small. The intensity of the 2.2-eV-peak component greatly depends on the amount of Zn2+, whereas those of the 2.0-eV-and 1.9-eV-peak components do not. Figure 4 plots the integrated areas of the sum and each of the three Gaussian components divided by the number of luciferin molecules, which are the quantum yield and contributions of the respective components, against the Zn2+ amount. It indicates that the contribution of the 2.2-eV-peak component decreases with the Zn2+ amount, whereas those of the 2.0-eV- and 1.9-eV-peak components are flat below 1 mM of Zn2+. The spectral shift, or color change, together with the decrease in quantum yield turns out to stem from the decrease in the 2.2-eV-peak component. Note that these features in Figs. 3 and 4 that are induced by the increased concentration of Zn2+ are very similar to those induced by decreased pH (10): The 2.2-eV-peak intensity was sensitive to pH as well as to the concentration of Zn2+ ions, while the 2.0-eV- and 1.9-eV-peak intensities were insensitive. Over 1 mM of Zn2+ inactivated luciferase so much that luciferin did not react completely, and this caused the drop in the intensities of all components. The spectral shift in the presence of Cd2+ had exactly the same characteristics as those in the presence of Zn2+. Three Gaussian components with the same peak energies as those of Zn2+ and Cd2+ were obtained in the quantitative analysis of bioluminescence spectra of samples with added Fe2+, Co2+ and Ni2+. However, unlike the individual intensity change of the 2.2-eV-peak component in the analysis of Zn2+ and Cd2+, the 2.0-eV-peak component slowly decreased with the addition of more Fe2+, Co2+ and Ni2+. This slow decrease was hardly noticeable without a quantitative analysis of the apparently similar spectral change to that of the samples with Zn2+ and Cd2+ ions. Regarding the influence of Hg2+ on the spectra, a pure red peak was observed as a result of adding only 0.1 mM of Hg2+. Because the bioluminescence weakened and the quantum yield was under 5%, it was impossible to do a precise analysis, as was done with Zn2+ and other transition metal ions. Figure 5 shows the integrated areas of the 2.2-eV-peak component divided by the number of luciferin molecules, or its contributions to the quantum yields, plotted against the concentration of various bivalent metal ions. Since the above analysis has shown that the bioluminescence color change is mainly caused by the change in the intensity of the 2.2-eV-peak component. Fig. 5 indicates the bioluminescence color sensitivity to various metal ions. The intensity decreases most rapidly in the presence of Hg2+, that is, the bioluminescence spectrum is most sensitive to Hg2+. The slopes are steeper for Zn2+ and Cd2+ than for Fe2+, Ni2+ and Co2+. The 2.2-eV-peak component in the presence of 1 mM of Zn2+ contributes 5% to the quantum yield, and the corresponding spectrum peaks at 620 nm and has an apparently symmetric shape, as is shown in Fig. 2. About 1 mM of Cd2+, or less than 0.02 mM of Hg2+, or more than 10 mM of Fe2+, Co2+ or Ni2+ are required to decrease the contribution of the 2.2-eV-peak component to the same level, e.g. 5%. Consider the slope difference of plots in Figs. 1 and 5, and the concentrations of metal ions required to decrease the quantum yield and the intensity of the 2.2-eV-peak component to half of their maximums (e.g. about 1 mM of Zn2+ or Cd2+, or about 4 mM of Ni2+, Co2+ or Fe2+ are required to reduce the quantum yield by half), we put quantum yield and spectral sensitivity of firefly bioluminescence in the following order, Hg2+ > Zn2+, Cd2+ > Ni2+, Co2+, Fe2+ [much greater than] Mg2+, Mn2+, Ca2+. Reaction rate Figure 6 shows the decay time constant of the bioluminescence intensity versus the concentration of various metal ions. The plot shows that for most of the metal ions higher concentration accelerates reaction, consistently with the usual chemical reaction kinetics. However, Ca2+ and Fe2+ are exceptions. The Ca2+ ion was such a poor catalyst that it took about 3-4 h to consume all the luciferin in our assays. Over 2 mM of Ca2+ decelerated the reaction so much that the reaction rate became too difficult to evaluate. More Fe2+ ions reduced the reaction rate because oxidation of bivalent Fe2+ into Fe3+ is a simultaneous competing reaction and a brownish precipitant was observed during the reaction. The reaction accelerated when more Hg2+ were present, but Hg2+ -ion data are not included in Fig. 6. because the bioluminescence intensity decay was not single exponential, which probably was due to the rapid inhibition of luciferase by Hg2+. Mn2+ ions were such active catalysts that they accelerated reactions even more effectively than Mg2+ ions. The dependence of the bioluminescence decay time on various metal ions shown in Fig. 6 has no definite correlation with those of the quantum yield and spectrum shown in Figs. 1 and 5. This indicates that the reaction rate is determined by a different mechanism from that of the quantum yield and spectrum. The rate of the reactions with over 1 mM of Zn2+, Cd2+ and Ni2+ ions is not included because luciferase was deficient in these cases. DISCUSSION One of the known roles of bivalent metal ions in firefly bioluminescence is binding to ATP to assist the conversion of luciferin to form the intermediate luciferyl-AMP and produce a pyrophosphate. The result that high concentration of metal ions accelerates reaction is consistent with the reaction model. Moreover, the order of binding affinity to ATP, Mn2+ > Mg2+, Zn2+ > Ca2+ (4,12), is analogous to the order of reaction rates with different kinds of bivalent metal ions in Fig. 6. Thus, it is reasonable to interpret that the difference of reaction rate with various kinds of metal ions stems from the luciferin-ATP-activation stage, where reaction rate is dominantly determined by the binding affinity of metal ions to ATP. On the other hand, the metal ions must have an effect on the bioluminescence spectral colors during the radioactive-decay stage of oxyluciferin, as in the case of the effect of pH. The issue of the color-change mechanism of the firefly bioluminescence with pH has long been investigated but it remains unsolved. The color-change features as a result of adding Zn2+ and Cd2+ are very similar to those due to pH change, and there is almost no color change resulting from added Mg2+, Mn2+, or Ca2+. Moreover, the order of spectral sensitivity is Hg2+ > Zn2+, Cd2+ > Ni2+, Co2+, Fe2+ [much greater than] Mg2+, Mn2+ and Ca2+, and these may be important clues to the mechanism. Because great differences in spectral sensitivities were found among the various bivalent metal ions in spite of their chemical similarities, and because the observed order of the spectral sensitivities has no correlation with the strength of the electron-withdrawing inductive effect of metal ions, it is unlikely that the color change originates from direct combination of metal ions with oxyluciferin. It is more likely that metal ions modify luciferase and the modified luciferase affects oxyluciferin so as to change the color of the bioluminescence. The strong difference and the peculiar order in spectral sensitivities for the various bivalent metal ions may stem from the special affinity of various residues in luciferase to those metal ions. It has been reported that substituting cysteine residue of luciferase with serine residue caused a loss in luciferase activity (13,14) and that the redshift in the spectra caused by CdCl2 could be partially reversed by adding excess cysteine (15); however, there was no report on the spectral change brought about by the substitution. We propose a possible interpretation whereby the quantum yield and spectral change in the presence of Zn2+ and Cd2+ obtained in our experiments comes from the binding of Zn2+ and Cd2+ to cysteine residues of luciferase. The binding alters the conformation of active sites of luciferase and thus affects the microenvironment for the excited state of oxyluciferin. The larger thiol binding affinity of Cd2+ than that of Zn2+ (16-18) may explain why the 2.2-eV-peak component is slightly more sensitive to Cd2+ than to Zn2+. Spectral change due to Fe2+, Ni2+ and Co2+ are qualitatively similar but quantitatively different from those of Zn2+ and Cd2+. By adding these three metal ions, the apparent color shift from yellow-green to red is caused by a fast decrease in the 2.2-eV-peak component and a slow decrease in the 2.0-eV-peak component. Therefore, we believe this different behavior from Zn2+ and Cd2+ may come from binding of these metal ions to different luciferase residues from the ones that Zn2+ and Cd2+ bind to. We assume that they bind to histidine residues of the protein (19,20). The finding that more Fe2+, Ni2+ and Co2+ ions than Zn2+ and Cd2+ ions are required to cause the same color shift in our experiment and the fact that P. pyralis luciferase has 14 histidine and four cysteine residues (21) are also consistent. The effects of metal ions on firefly bioluminescence (2-4) were previously studied by measuring the initial activities or bioluminescence intensities just after mixing the solutions. However, the activities reflect both reaction rate and quantum yield, and the effects of metal ions on the reaction rate and quantum yield turned out to be independent in the present study. Therefore, separate measurements on reaction rates, quantum yields and spectra are indispensable for gaining a basic understanding of firefly bioluminescence. The change in bioluminescence from yellow-green to red in the presence of bivalent metal ions such as Zn2+ and Cd2+ ions were first reported by Seliger et al. in 1964 (2). The bioluminescence spectral data were shown as normalized spectra, and the color change was regarded as an intensity conversion from a green peak to a red peak, as was the case with the color change due to pH change (5). However, our present experiments have shown that the 2.2-eV-peak intensity that was sensitive to pH was also sensitive to the concentrations of Zn2+ and Cd2+ ions, while the pH-insensitive 2.0-eV-and 1.9-eV- peaks were also insensitive to these metal ions. Therefore, we find that the key to understanding the color-change mechanism with pH and metal ions may be the high sensitivity of the green 2.2-eV-peak intensity. CONCLUSIONS We quantitatively investigated the influence of bivalent metal ions on quantum yield, spectrum and reaction rate of firefly bioluminescence. Our experiments yielded new knowledge on the influence of metal ions on the activity of luciferase, which is usually evaluated in terms of the initial bioluminescence intensity. Increasing the amount of Mn2+ and Mg2+ ions did not change the quantum yield or color of firefly bioluminescence but rather accelerated the reaction. This acceleration will display an increase in reactivity in traditional evaluation methods. On the other hand, the apparent inhibition due to other metal ions can be divided into four types: the one caused by Ca2+ ion does not change the quantum yield or spectrum; it only decelerates the reaction; the second one caused by Hg2+ has the characteristics of dramatic decrease in quantum yield and rapid redshift of spectral peak; the third one, as represented by a relatively rapid color shift to red, is caused by Zn2+ and Cd2+ ions; and the last kind of hindrance is caused by Fe2+, Ni2+ and Co2+ ions and is characterized as a similar color shift but one that is less sensitive to the amount of metal ions in comparison with the third type of change. In analytical applications such as gene reporting, these effects of metal ions must be taken into account because many of these bivalent metal ions exist or coexist in living cells. In such a case, the intensity of the detected signal will be determined by the amount of luciferase and its activity under the influence of metal ions. Our results are a valuable reference for quantitative evaluations in firefly bioluminescence applications. We learned that the color change of firefly bioluminescence in the presence of various amounts of bivalent metal ions such as Zn2+ shows equivalent quantitative characteristics to the change under various pH reported by Y. Ando et al. (10). This similarity must be taken into consideration when studying the color-determining mechanism of firefly bioluminescence. We also find that in spite of the extremely similar spectral change, the bioluminescence spectra under the influence of Fe2+, Ni2+ and Co2+ ions behave differently from those under the influence of Zn2+ and Cd2+ ions. To explain the color change, we should consider not only the pH effect but also contribution of these bivalent metal ions. Our experimental data provide some clues for theoretical analyses such as quantum chemical calculations on the color-change mechanism. Acknowledgements-This research was partly supported by KAKENHI (20360135) of JSPS, and the Photon Frontier Network Program of MEXT, Japan. One of us (Y.W.) is also grateful for support by International Priority Graduate Programs of MEXT, Japan. REFERENCES 1. Roda, A., P. Pasini, M. Mirasoli, E. Michelini and M. Guardigli (2004) Biotechnological applications of bioluminescence and chemiluminescence. Trends Biotechnol. 22, 295-303. 2. Seliger, H. H. and W. D. McElroy (1964) The colors of firefly bioluminescence: Enzyme configuration and species specificity. Proc. Natl Acad. Sci. USA 52, 75-81. 3. Morton, R. A., T. A. Hopkins and H. H. Seliger (1969) The spectroscopic properties of firefly luciferin and related compounds. An approach to product emission. Biochemistry 8(4), 1598-1607. 4. Lee, R. T., J. L. Denburg and W. D. McElroy (1970) Substrate-binding properties of firefly luciferase II. ATP-binding site. Arch. Biochem. Biophys. 141, 38-52. 5. Seliger, H. H. and W. D. 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Inhibition by metal ions and activation by amino acids and other chelating agents. J. Bio. Chem. 256, 11160-11165. 20. Stohs, S. J. and D. Bagchi (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321-336. 21. Conti, E., N. P. Franks and P. Brick (1996) Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4, 287-298. Yu Wang*1, Hidehiro Kubota2, Nobuyuki Yamada2, Tsutomu Irie2 and Hidefumi Akiyama1 1 Institute for Solid State Physics, University of Tokyo, Kashiwa, Chiba, Japan 2 ATTO Corporation, Bunkyo-ku, Tokyo, Japan Received 19 December 2010, accepted 28 March 2011, DOI: 10.1111/j.1751-1097.2011.00931.x * Corresponding author email: [email protected] (Yu Wang) © 2011 The Authors Photochemistry and Photobiology © 2011 The American Society of Photobiology 0031-8655/11 (c) 2011 American Society for Photobiology [ Back To TMCnet.com's Homepage ]