J. Phys. Chem. B 2010, 114, 12679–12684
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Spectroscopic Properties of Curcumin: Orientation of Transition Moments Anindita Mukerjee,‡ Thomas J. Sørensen,§ Amalendu P. Ranjan,‡ Sangram Raut,† Ignacy Gryczynski,†,‡ Jamboor K. Vishwanatha,‡ and Zygmunt Gryczynski*,†,‡ Center for Commercialization of Fluorescence Technologies and Department of Molecular Biology and Immunology, UniVersity of North Texas Health Science Center, 3500 Camp Bowie BouleVard, Fort Worth, Texas 76107, and Nano-Science Center and Department of Chemistry, UniVersity of Copenhagen, UniVersitetsparken 5, DK-2100 KøbenhaVn Ø, Denmark ReceiVed: May 4, 2010; ReVised Manuscript ReceiVed: June 28, 2010
Curcumin, a naturally occurring yellow-orange pigment with potent antioxidant and antitumor properties, has been attracting researchers from a wide range of fields including chemistry, spectroscopy, biology, and medicine. Ultrafast excited-state processes such as solvation and excited-state intramolecular hydrogen atom transfer (ESIHT) make curcumin an attractive agent for photodynamic therapy. In this report we present studies of linear dichroism and fluorescence anisotropy in oriented and isotropic media. The results show transition moments (long wavelength absorption and emission) oriented along the long molecular axis. Comparison of linear dichroism and excitation anisotropy in oriented and isotropic media suggests that excitedstate intramolecular hydrogen atom transfer is probably associated with intramolecular conformational changes that can be constrained in highly stretched poly(vinyl alcohol) (PVA) film. 1. Introduction Curcumin is a naturally occurring yellow-orange pigment found in the rhizomes of Curcuma longa (turmeric). Research over the last few decades has shown that curcumin exhibits significant antioxidant and anti-inflammatory properties that facilitate wound healing, along with strong therapeutic potential against a variety of cancers while being pharmacologically safe even at high doses.1-5 Curcumin, as a potent antioxidant, has been shown to be a potent scavenger of a variety of reactive oxygen species including superoxide anion radicals, hydroxyl radicals,6 and nitrogen dioxide radicals,7 thus playing a major role in the possible treatment of oxidative-stress-related diseases. Curcumin has been shown to suppress transformation, proliferation, and metastasis of tumors. These effects are mediated through its regulation of various transcription factors, growth factors, inflammatory cytokines, protein kinases, and other enzymes. The compound also inhibits proliferation of cancer cells by arresting them in various phases of the cell cycle and by inducing apoptosis. Curcumin has been shown to have dosedependent chemopreventive and therapeutic effects against cancers of the blood, skin, oral cavity, lung, pancreas, and intestinal tract, and to suppress angiogenesis and metastasis in rodents.8 Due to its photochemical properties, curcumin has been shown to have a great potential to be an effective photodynamic agent.9-14 Spectral properties of curcumin may potentially enable optical imaging/monitoring many of its physiological functions. Multiple studies have focused on using steady-state and timeresolved fluorescence spectroscopy to investigate the photophysical and biochemical properties of curcumin in various solvents. Studies of excited-state photophysics of curcumin, on * Author for correspondence. E-mail:
[email protected]. † Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center. ‡ Department of Molecular Biology and Immunology, University of North Texas Health Science Center. § University of Copenhagen.
the subnanosecond time scale, have provided some fundamental understanding of excited-state intramolecular hydrogen atom transfer (ESIHT) and its role in biomedicinal functions.15,16 The ultrafast hydrogen atom transfer and associated conformational changes may play an important role in medicinal properties of curcumin. Recent developments of curcumin-loaded nanoparticles present an interesting drug delivery platform and/or imaging agent.17 Understanding the physicochemical properties of curcumin embedded in a solid matrix (biodegradable polymer beads) is important for further development of such approaches.17 To better understand the physicochemical basis of interaction of curcumin with other biological/chemical molecules, it is necessary to study its fundamental spectroscopic and physicochemical properties. In this report, we present detailed studies of polarized absorption in oriented poly(vinyl alcohol) (PVA) films (linear dichroism), steady-state, time-resolved fluorescence and fluorescence polarization of curcumin in various solvent, isotropic and in oriented PVA films. The purpose was to determine the absorption and emission transition moments orientations in curcumin molecule as well as to investigate the effect of various solvents on excited-state deactivation. Comparison of linear dichroism data with excitation anisotropy in oriented polymer films confirms the idea of ultrafast conformational processes associated with two tautomeric forms of curcumin.15 The information on transition moments orientation and their relative contribution to ultrafast deactivation processes will be crucial for the interpretation of fluorescence polarization data and for the future studies that will involve Fo¨rster resonance energy transfer (FRET) between curcumin molecules (homoFRET) and curcumin and other suitable dyes that can serve as an excitation energy donor or energy acceptor.18,19 2. Basic Theory Before presenting the studies of transition moments and emission polarization in isotropic and axially oriented systems,
10.1021/jp104075f 2010 American Chemical Society Published on Web 09/14/2010
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we need to introduce necessary nomenclature traditionally used to interpret the results. To determine the directions of electronic transition moments of the chromophore molecule one should first orient molecules in a controllable fashion. From many various techniques used for molecular orientation,20,21 an axially stretched polymer such as poly(vinyl alcohol) (PVA) is the simplest and most effective way. An orientation of luminescent molecules directly depends on the polymer stretch ratio Rs defined as the axial ratio a/b of an ellipse (a and b are the semi-major and -minor axes) deformed from a circle of radius r initially drawn on the film.20,22 The Rs value directly relates to the ratio of polymer length before and after stretching, R as Rs ) R3/2. Spectroscopic measurements with polarized light have been extensively developed for systems with anisotropic distribution of the molecular transition moments in oriented media. In such cases, a dependence of the absorption on the orientation of the polarization plane of the incident light with respect to a given direction of the sample orientation has been observed.23-25 A number of different definitions for observed absorption irregularity such as dichroic ratio (d ) A|/A⊥) have been defined. Originally Pringsheim23 defined dichroism, D as a measure of system anisotropy:
D)
A|| - A⊥ d-1 ) A|| + A⊥ d+1
(1)
r)
A|| - A⊥ A|| - A⊥ A|| - A⊥ ) ) and K ) 1/3(A|| + 2A⊥) 1/3A A|| + 2A⊥ A|| - A⊥ (2) A
A ) A| + 2A⊥ is the total absorbance, and the isotropic absorbance is Aiso ) 1/3A. The absorption anisotropy K is related to the reduced linear dichroism by LDr ) 3K and to the dichroic ratio by Rd ) (2K + 1)/(1 - K). 2.1. Fluorescence Anisotropy (r). For fluorescent molecules in the system that has cylindrical symmetry (like a population of excited molecules generated by excitation with linearly polarized light), emission anisotropy is defined:28,29
(
)
(4)
A general property of fluorescent molecules, according to which the equilibrated lowest excited states and consequent emission spectrum are independent of the excitation wavelength, makes possible the resolution of the polarization of the overlapping absorption bands.18,19,26 In this case of overlapping transitions, fluorescence anisotropy can be expressed in terms of weighted sums of the anisotropies belonging to the different contributing transitions:
jr )
∑ firi
(5)
i
where ri is the anisotropy fraction associated with the ith absorption transition in the molecule, and fi is the normalized contribution (probability) of the ith transition moment to the total absorption at the given excitation wavelength. 2.2. Fluorescence Lifetimes. The lifetime of the excited state (fluorescence lifetime, τ) is the average time a molecule spends in the excited state prior to returning to the ground state. After excitation, the deactivation process is governed by the emissive rate constant Γ and nonradiative rate constant knr that depopulate the excited state, leading to the relationship describing fluorescence lifetime:
To generalize this definition by relating it to the overall isotropic or total absorption, the concepts of reduced linear dichroism, LDr,24,25 and absorption anisotropy, K,26,27 have been introduced:
LDr )
2 3 1 cos2 β 5 2 2
τ)
1 Γ + knr
(6)
The fluorescence lifetime strongly depends on changes in the rate constants. Typically interactions with the environment such as solvent or other solutes may lead to changes in the nonradiative rate, knr, resulting in a solvent-dependent lifetime change. The change in fluorescence lifetime very well reflects environmental changes and is commonly used for monitoring inter- and intramolecular interactions. Time-dependent fluorescence intensity decays are typically described by the exponential form where multiexponential decay can be presented as:
τ)
∑ Ri et(Γ+k ) ) ∑ Ri et/τ nr
i
(7)
where Ri is the fractional amplitude of the ith fluorescence lifetime component.18,19 3. Materials and Methods
I|| - I⊥ r) I|| + 2I⊥
(3)
where I| and I⊥ are the components of fluorescence intensity polarized parallel and perpendicular to the direction of the electric vector of the excitation light. The term I| + 2I⊥, represents the total fluorescence intensity. In general, in the absence of depolarization factors (rotational diffusion or excitation energy migration), the value of r directly relates to the angle β between absorption and emission transition moments in the molecules.18,19 For separated transitions, fluorescence anisotropy can be expressed as:
Curcumin (total curcuminoids content: 96.85%; curcumin content: 76.07%) was obtained from Sabinsa Corporation (NJ, U.S.A.). Curcumin was diluted in ethanol at different concentration (25, 50, 100, 250 µg/mL) prepared according to literature procedures. 3.1. Film Preparation. PVA films were prepared from 12% aqueous solution. The curcumin dissolved in ethanol was added to the prewarmed PVA solution. The PVA solution containing curcumin was poured into Petri dishes and dried.25,26 PVA films were prepared and stretched in the conditions previously described.25,26 Films used for fluorescence anisotropy measurements contained lower concentrations of curcumin so as to compare to films used for absorption.
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Figure 1. Schematic of front-face configuration for anisotropy measurements in thin films.
3.2. Experimental Methods. Linear dichroism was measured using a Varian DS50 spectrophotometer equipped with a homebuilt adapter for a polymer film sample holder. A polarizer was placed in the front of the oriented sample. The sample can be positioned parallel or perpendicular to the light polarization in respect to the sample orientation (stretching direction) as earlier described.26,27 The absorption of the sample was corrected for a baseline obtained with the blank PVA films and then corrected for the film absorption calibrated for the thickness.20,27 Polarized fluorescence measurements of curcumin in PVA films were performed in a specially adapted PC1 module (ISS Inc.) as shown in Figure 1. In place of the regular square geometry adapter, we introduced two mirrors in the excitation line so as to form a front face excitation of the film mounted in a homemade holder in the center of the sample compartment of the PC1 module. To avoid any depolarization effects at the film surfaces, the film was placed orthogonally to the direction of observation. Correction factors in the range of 450-650 nm were obtained by measuring methanol solutions of fluorescein and azadioxatriangulenium in a 0.2 mm cuvette mounted in the place of the film. Both fluorescein, (fluorescent lifetime, τav ) 4 ns) and azadioxatriangulenium, (τ ≈ 20 ns) at 25 °C in nonviscose methanol, have a very short rotational correlation time and were used as the zero anisotropy standards for steady-state emission. Polarized fluorescence measurements of curcumin in solvents (ethanol, water, glycerol) were done using PC1 and/or Varian Eclipse fluorometers in a standard 10 mm × 4 mm cuvette with right-angle square geometry optics (short path used for excitation). Time-resolved fluorescence intensity decays were measured using the FT200 (Picoquant GmbH) system equipped with a multichannel plate photomultiplier (MCP PMT) detector. For excitation, we used a 470 nm pulsed laser diode with the pulse width of about 50 ps. The instrument response was specially calibrated for optical delays as described by us,30 and the expected resolution after pulse deconvolution was better than 10 ps.
water (pH ) 7.2), ethanol, glycerol, and in the isotropic PVA film. Intrinsically, the absorption spectra in water and other solvents are broader than absorption spectrum in the PVA film. Also, the absorption spectrum in rigid PVA matrix is shifted toward the longer wavelength. In contrast, the emission spectrum in PVA is shifted to the shorter wavelengths and is narrower than in other solvents. This may indicate that curcumin in water/ solvents adopts multiple conformations as a result of polar interactions. In turn, the absorption spectrum in PVA film is narrower and slightly asymmetrical probably indicating lower number of possible conformers. 4.2. Linear Dichroism. The parallel (A|) and perpendicular (A⊥) absorption components of curcumin in 5-fold stretched PVA films (Rs ) 11.2) are shown in Figure 3. The corresponding absorption anisotropy (K) is also shown in the figure. Notably, the parallel and perpendicular absorption components are distinctly different, leading to very pronounced absorption anisotropy (K) dependence. High value of absorption anisotropy for the long wavelength absorption indicates that absorption
4. Results Scheme 1 shows the curcumin molecule where long axis corresponds to the preferential orientation in the stretched polymer films. 4.1. Absorption Spectra. Graphs A and B of Figure 2 show the normalized absorption and emission spectra of curcumin in
Figure 2. (A) Normalized absorption spectra of curcumin in water, ethanol, glycerol and isotropic PVA film. (B) Normalized emission spectra of curcumin in water, ethanol, glycerol, and isotropic PVA film (excitation wavelength: 430 nm).
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Figure 3. Parallel and perpendicular components of absorption, and absorption anisotropy of curcumin in 5-fold stretched PVA films (Rs ) 11.2).
Figure 4. (A) Excitation anisotropy of curcumin in water, ethanol, glycerol, and isotropic PVA films. (B) Emission anisotropy of curcumin in water, ethanol, glycerol at 20 °C and isotropic PVA films (excitation wavelength: 430 nm).
transition moment is oriented along the long molecular axis (preferential orientation axis). At shorter wavelengths, the absorption anisotropy quickly declines to the value below 0.3. This indicates significant contribution of transitions that have orientation orthogonal to the long molecular axis. 4.3. Excitation and Emission Anisotropies. Graphs A and B of Figure 4 respectively show excitation and emission anisotropies in various solvents and in isotropic PVA film. The excitation anisotropies were collected at observation wavelength
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Figure 5. Excitation anisotropy of curcumin in isotropic (unstretched) PVA film, 2-fold stretched film, 4-fold stretched film. The simulated excitation anisotropy and the normalized absorption are included for comparison.
550 nm for water and glycerol and 530 nm for ethanol (all spectra were measured at 20 °C). Excitation anisotropy for isotropic PVA films was measured at 500 nm observation wavelength. For comparison the anisotropy spectra should be related to absorption and emission spectra shown in A and B of Figure 2. The excitation anisotropy spectra have distinct wavelength dependence. The excitation anisotropy quickly drops for wavelengths below 400 nm. This is consistent with linear dichroism measurements shown in Figure 3. A strongly decreasing anisotropy for shorter excitation wavelengths confirms that in the region below 400 nm, significant orthogonal transitions contribute to curcumin absorption. Emission anisotropies in viscose glycerol or rigid PVA film are very high (r > 0.35). Relatively high steady-state anisotropy in low viscosity ethanol and water are due to short fluorescence lifetime of curcumin. A constant emission anisotropy value across the emission wavelengths indicates a single emission transition or multiple transitions oriented along the long molecular axis. Figure 5 shows the excitation anisotropy of curcumin in isotropic and stretched PVA films. The anisotropy dependence for isotropic film (also shown in Figure 4) is similar to that in glycerol at 20 °C and significantly decreases for shorter wavelengths. Typically one would expect that orientating molecules (stretching the film) will increase the difference between anisotropies observed for long and short excitation wavelengths. But intrinsically, the excitation anisotropy dependence becomes more flat as the PVA film is stretched. A 4-fold stretched film shows very high, long wavelength anisotropy (∼0.9), indicating excellent alignment of curcumin molecules in the film. However, as the film stretching progresses, the decrease of excitation anisotropy at shorter wavelengths becomes smaller (practically negligible). For high stretching ratios, the anisotropy dependence is almost flat, indicting that only transitions with directions along the molecular axis contribute to the emission. This is unexpected and indicates that immobilization of curcumin in highly stretched polymer prevents some intramolecular processes. For comparison we indicated by solid line the expected wavelength-dependent excitation anisotropy for 4-fold stretched PVA film. The difference between expected and measured anisotropies is striking. 4.4. Fluorescence Lifetimes. In Figure 6, we present fluorescence intensity decays measured for curcumin solution in ethanol, water, glycerol, and isotropic PVA film (film measurements were done at front-face configuration). The recovered
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Figure 6. Fluorescence intensity decays measured for curcumin solution in ethanol, water, glycerol, and isotropic PVA film (film measurements were done in front-face configuration).
TABLE 1: Recovered Fluorescence Lifetimes in Ethanol, Water, Glycerol, and Isotropic PVA Film
a
solvent
τav (nsa)
ethanol water glycerol isotropic PVA film
0.12 0.13 0.51 0.99
ns - nanoseconds.
fluorescence lifetimes are presented in Table 1. In ethanol and water, fluorescence decays can be sufficiently fitted with two exponential decays with a dominant, very short component. The average fluorescence lifetime in ethanol at 20 °C is 120 ps, and in water it is 130 ps. Interestingly for glycerol, the intensity decay is more heterogenic and the lifetime components are much longer. Resulting average fluorescence lifetime in glycerol is about 510 ps. In PVA film, two long lifetime components are measured giving average lifetime of 990 ps. This clearly indicates a better stabilization of the curcumin excited state in the PVA matrix. 5. Conclusions Linear dichroism and emission polarization studies show that transition moments for the long wavelength absorption band and emission band are oriented along the long molecular axis of the curcumin molecule. High and constant anisotropy across the emission band suggests that the emission transition is a single transition. The absorption anisotropy at shorter wavelengths (below 400 nm) is much lower, indicating the presence of orthogonal transitions as compared to orientation axis. Decrease of excitation anisotropy at shorter excitation wavelengths in isotropic PVA film is consistent with linear dichroism measurements. In fact, wavelength-dependent excitation anisotropy in various solvents follows a similar pattern, and that found in high-viscosity glycerol is very similar to that in isotropic PVA. Decrease of linear dichroism and corresponding decrease of excitation anisotropy at shorter wavelengths in isotropic solutions indicate that excited orthogonal states contribute to the curcumin emission. Progressive stretching of the PVA film results in proportional increase of wavelength-dependent absorption anisotropy (linear dichroism). However, as the film stretch-
ing progresses, the anisotropy at shorter wavelengths increases faster, and thus, the short wavelength anisotropy decrease becomes less evident. For highly stretched film the short wavelength effect almost vanishes completely. This is a rather surprising observation since it is well accepted that PVA film does not affect molecular properties of typical dyes. We tested if this effect depends on curcumin concentration. The excitation energy migration31 may change observed anisotropy; however, in our case, we did not observe any significant concentration dependence. The origin of short wavelength behavior is not clear, and we can only suggest one possible explanation. As the polymer stretches, dye molecules are tightly packed between PVA chains. This is a mechanism for molecular orientation. Adhikary et al.15 proposed a mechanism of excited-state intramolecular hydrogen atom transfer (ESIHT) that plays an important role in excited-state photophysics of curcumin. Indeed, our studies in various viscosity solvents (ethanol, water, glycerol) also confirm that the ESIHT is insensitive to solvent viscosity. Also, in isotropic PVA (a solid matrix) the ESIHT is comparable to that in high viscosity glycerol. But in highly stretched PVA film, we do not observe ESIHT affecting the excitation anisotropy. A possible explanation could be that the curcumin molecules exist as two tautomeric forms.15 The difference in the direction of transition moment in two different tautomeric forms can be detected by linear dichroism as we already demonstrated for 5-phenyltetrazole.32 The dominant form in the ground state is keto-enol tautomer. Excitation of curcumin leads to breakage of the hydrogen bond and allows small conformational change (probably a cis-trans conformational change of the enol double bond). Similar to transstilbenes, one form has better fluorescence.33 One of the tautomers is probably preferentially excited at shorter wavelength and has transition direction orthogonal to long molecular axis. The conformational change associated with the transition from one form to another is small and happens very quickly even in very viscous solvents. As shown by Adhikary et al.,15 the time constant is ∼70 ps in methanol and longer (∼120 ps) in ethylene glycol. Our time-resolved measurements indicate comparable curcumin fluorescence lifetimes in water and ethanol and much longer in glycerol and PVA films. Evidently in isotropic polymer film, there is enough space for small conformational switching to occur, and the excitation anisotropy behavior is very similar to that of glycerol. Nevertheless, the conformational change in viscous (rigid) solvents is probably slower, and observed fluorescence lifetimes in the PVA film are longer than in glycerol. Stretching the polymer film aligns curcumin molecules and packs them tightly within the polymer chains preventing the tautomerization or more probably conformational transition. However, only one form makes a dominant contribution to the emission. Stretching the polymer does not change equilibrium between tautomeric forms, and both forms are still detectable by absorption (linear dichroism) in the highly stretched films. Acknowledgment. Curcumin was obtained from Sabinsa Corporation through a license agreement between SignPath Pharmaceuticals Inc., PA, U.S.A. and UNTHSC (J.K.V.). This work was supported in part by a Sponsored Research Agreement with SignPath Pharmaceuticals Inc, PA, U.S.A. (J.K.V.), DOD Grant W81XWH-09-1-0406 (Z.G.) and by Texas ETF (C.C.F.T.). References and Notes (1) Anand, P.; Sundaram, C.; Jhurani, S.; Kunnumakkara, A. B.; Aggarwal, B. B. Cancer Lett. 2008, 267, 133–164.
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