Fluorescence Polarization Studies Research Articles

Manipulation of Spontaneous Emission Dynamics of Organic Dyes in the Porous Silicon Matrix

The control of the spontaneous emission (SE) rate of dye molecules (4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) and Coumarin 523 (C523)) embedded in the Porous Silicon (PS) matrix has been studied using picosecond resolved fluorescence decay and polarization studies. We have shown that the SE rates of the two organic dyes embedded in the PS matrix depend on the relative positions of the emission maxima of the dyes with respect to electronic band gap energy of the PS matrix. We have also explored that the electronic band gap of the host PS matrix can easily be tuned by partial oxidation of the PS and the nature of SE of the embedded dyes can be tuned accordingly. The demonstrated retardation or enhancement of the spontaneous photon emission may enable the application of fluorescent organic molecules in PS matrix in several quantum optical devices including the realization of single photon sources.

Organic Osmolyte Mediated Kinetic Capillary Electrophoresis for Study of Protein−DNA Interactions

An organic osmolyte mediated kinetic capillary electrophoresis is developed and applied to study the binding of Escherichia coli RecA protein to single-stranded (ss) DNA. The complex of RecA and ssDNA can be significantly enhanced and stabilized by 20% glycerol over 5.6 times. Kinetic capillary electrophoresis study reveals that glycerol may function in the preservation of RecA native conformation, enhancement of specific binding affinity (20 times), and decrease in dissociation rate constant (1 time). The combined laser-induced fluorescence polarization (LIFP) study further indicates a tightened assembly of RecA on ssDNA induced by glycerol. The binding of RecA to ssDNA was also enhanced by two additional protecting organic osmolytes, sucrose and PEG 400. In contrast, a denaturing organic osmolyte, urea, even at a concentration of 110 mM can prevent the formation of the RecA-ssDNA complexes. The detection of the binding of E. coli single-stranded binding protein (SSBP) to ssDNA was also improved by the same method. By combining the protection of organic osmolytes with low-temperature storage, a method for preservation of protein-DNA complex up to 43 h is further demonstrated. By use of a long capillary and protecting glycerol together an enhanced kinetic CE analysis was also developed, providing a better separation and analysis of the undissociated complex, the dissociated complex, and the unbound probe. The results demonstrate the wide applicability of the method to the study of various protein-DNA binding.

Conformation and Dynamics of a Rhodamine Probe Attached at Two Sites on a Protein: Implications for Molecular Structure Determination in situ

Replica exchange molecular dynamics (REMD) calculations were used to determine the conformation and dynamics of bifunctional rhodamine probes attached to pairs of cysteines in three model systems: (a) a polyalanine helix, (b) the isolated C helix (residues 53-66) of troponin C, and (c) the C helix of the N-terminal region (residues 1-90) of troponin C (sNTnC). In each case, and for both diastereoisomers of each probe-protein complex, the hydrophobic face of the probe is close to the protein surface, and its carboxylate group is highly solvated. The visible-range fluorescence dipole of the probe is approximately parallel to the line joining the two cysteine residues, as assumed in previous in situ fluorescence polarization studies. The independent rotational motion of the probe with respect to the protein on the nanosecond time scale is highly restricted, in agreement with data from fluorescence polarization and NMR relaxation studies. The detailed interaction of the probe with the protein surface depends on steric factors, electrostatic and hydrophobic interactions, hydrogen bonds, and hydration effects. The interaction is markedly different between diastereoisomers, and multiple preferred conformations exist for a single diasteroisomer. These results show that the combination of the hydrophobic xanthylium moiety of bifunctional rhodamine with the carboxylate substitution in its pendant phenyl ring causes the probe to be immobilized on the protein surface, while the two-site cysteine attachment defines the orientation of its fluorescence dipole. These features allow the orientation of protein components to be accurately determined in situ by polarized fluorescence measurements from bifunctional rhodamine probes.

Photodynamic therapy with intratumoral administration of Lipid‐Based mTHPC in a model of breast cancer recurrence

Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer. Mice were directly injected into the tumor (IT) with 25 microl of a Foslip suspension (0.15 mg/ml) and illumination (652 nm, 20 J/cm(2)) was performed at different time points with pathological assessment after 48 hours. In a parallel mice series plasma samples were obtained at different endpoints after IT Foslip injection for HPLC analysis and the tumors were subjected in toto to macrofluorescence imaging. Fluorescence polarization measurements were conducted in vitro to estimate the rate of sensitizer redistribution from liposomes. Optimal, albeit partial, cure rates were obtained at 24 hours post-sensitizer and uninistration. Inhomogeneous and weak fluorescence was observed at early time points and became maximal at 24 hours. Plasma levels of mTHPC increased until 15 hours. Fluorescence polarization measurements showed a slow sensitizer transfer from liposomes to model membranes. The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response.

Ethylene signaling: Identification of a putative ETR1–AHP1 phosphorelay complex by fluorescence spectroscopy

The plant ethylene receptor ETR1, which shows substantial sequence homology to typical bacterial histidine kinases, is involved in the coordination of several growth and development processes. Fluorescence polarization studies presented here demonstrate a specific interaction of ETR1 with the histidine-containing transfer protein AHP1, supporting the idea that a phosphorelay module is involved in ethylene signaling. The sensitive assay employed in our studies allows analysis of protein–protein interactions in a homogenous aqueous environment, exact control of external parameters, and quantitative analysis of the affinity and stability of the complex. Thereby it provides the basics for a more quantitative elucidation of phosphorelay modules acquired in phytohormone signaling.

Selective toxicity of ginsenoside Rg3 on multidrug resistant cells by membrane fluidity modulation

Multidrug resistance (MDR) is a major problem in cancer chemotherapy. It was previously reported that a red ginseng saponin, 20(S)-ginsenoside Rg3 could modulate MDR in vitro and extend the survival of mice implanted with ADR-resistant murine leukemia P388 cells. This study examined the cytotoxicity of Rg3 on normal and transformed cells, along with its effect on the membrane fluidity. The cytotoxicity study revealed that 120 microM of Rg3 was cytotoxic against a multidrug-resistant human fibroblast carcinoma cell line, KB V20C, but not against normal WI 38 cells in vitro. Flow cytometric analysis using rhodamine 123 as the artificial substrate showed that Rg3 promoted the accumulation of rhodamine 123 in ADR-resistant murine leukemia P388 cells in vivo. Fluorescence polarization studies using the hydrophilic fluorescent probe, DPH, and hydrophobic probe, TMA-DPH, showed that 20 microM Rg3 induced a significant increase in fluorescence anisotropy in KB V20C cells but not in the parental KB cells. These results clearly show that Rg3 decreases the membrane fluidity thereby blocking drug efflux.

Cyclooxygenase-2 Inhibition by Etoricoxib Modulates Plasma Membrane Fluidity in Rat Colon

The present study was designed to investigate the effects of a selective cycloogenase-2 (COX-2) inhibitor, etoricoxib, on the membrane-specific enzymes, lipid composition, and changes in fluidity parameters of rat colonic plasma membrane. Two doses of the drug were used, one within its therapeutic anti-inflammatory range as based on the ED50 value in rats (Eto-1), while the other at 10 times higher dose relating to the toxicity studies (Eto-2), which have not been reported so far. The activity of the membrane alkaline phosphatase was found to be increased after treatment with both the doses of etoricoxib as compared to the control. The total lipid and the cholesterol content showed a decrease while an increase in ganglioside sialic acid content was noted. Phospholipid content and the cholesterol-phospholipid molar ratio showed no change in either of the treatment groups. Fluorescence polarization studies with diphenylhexatriene showed that membrane fluidity was least altered in the isolated brush border membrane from colon or in the liposomes prepared from the membrane lipid extracts. Also, the translational diffusion studied with pyrene showed that the fluidity parameter was decreased as measured by the excimer formation. It is concluded that etoricoxib, a new generation COX-2 inhibitor (called the coxibs), appeared to be a safe nonsteroidal anti-inflammatory drug (NSAID) in the colonic membranes.

2P-046 1分子蛍光偏光法による古細菌型シャペロニンの構造変化解析(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

2P-046 1分子蛍光偏光法による古細菌型シャペロニンの構造変化解析(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

Novel N,N ′-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery - synthesis, in vitro transfection activity, and physicochemical characterization

Novel N,N'-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids were synthesized and their physicochemical properties in lamellar assemblies with and without plasmid DNA were evaluated to elucidate the structural requirements of these double-chained pH-sensitive surfactants for potent non-viral gene delivery and expression. The highest in vitro transfection efficacies were induced at +/-4:1 by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion of helper lipids. Transfection activities were reduced in the presence of either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained reporter gene expression levels at +/-4:1 and yielded increased lipofection activity at a lower charge ratio of +/-2:1. Ethidium bromide displacement indicated efficient plasmid DNA binding and compaction by the transfection-competent analogs. Dynamic light-scattering and electrophoretic mobility studies revealed lipoplexes of the active lipids with large particle sizes (mean diameter>or=500 nm) and zeta potentials with positive values (low ionic strength) or below neutrality (high ionic strength). Langmuir film balance studies showed high in-plane elasticity of these derivatives in isolation. In agreement with the monolayer experiments, fluorescence polarization studies verified the fluid nature of the highly transfection-efficient amphiphiles, with gel-to-liquid crystalline phase transitions below physiological temperature. The active compounds also interacted with endosome-mimicking vesicles to a greater extent than the poorly active derivative 1,3lb4, as revealed by fluorescence resonance energy transfer experiments. Taken together, the results suggest that well-hydrated and highly elastic cationic lipids with increased acyl chain fluidity and minimal cytotoxicity elicit high transfection activity.

Identification of the MxiH Needle Protein Residues Responsible for Anchoring Invasion Plasmid Antigen D to the Type III Secretion Needle Tip

The pathogenesis of Shigella flexneri requires a functional type III secretion apparatus to serve as a conduit for injecting host-altering effector proteins into the membrane and cytoplasm of the targeted cell. The type III secretion apparatus is composed of a basal body and an exposed needle that is an extended polymer of MxiH with a 2.0-nm inner channel. Invasion plasmid antigen D (IpaD) resides at the tip of the needle to control type III secretion. The atomic structures of MxiH and IpaD have been solved. MxiH (8.3 kDa) is a helix-turn-helix, whereas IpaD (36.6 kDa) has a dumbbell shape with two globular domains flanking a central coiled-coil that stabilizes the protein. These structures alone, however, have not been sufficient to produce a workable in silico model by which IpaD docks at the needle tip. Thus, the work presented here provides an initial step in understanding this important protein-protein interaction. We have identified key MxiH residues located in its PSNP loop and the contiguous surface that uniquely contribute to the formation of the IpaD-needle interface as determined by NMR chemical shift mapping. Mutation of Asn-43, Leu-47, and Tyr-50 residues severely affects the stable maintenance of IpaD at the Shigella surface and thus compromises the invasive phenotype of S. flexneri. Other residues could be mutated to give rise to intermediate phenotypes, suggesting they have a role in tip complex stabilization while not being essential for tip complex formation. Initial in vitro fluorescence polarization studies confirmed that specific amino acid changes adversely affect the MxiH-IpaD interaction. Meanwhile, none of the mutations appeared to have a negative effect on the MxiH-MxiH interactions required for efficient needle assembly.

Spectroscopic Characterization of Hydroxylated Nanoballs in Methanol

In this report, the photophysical properties of self-assembled [Cu(2)(5-OH-bdc)(2)L(2)](12) [where (5-OH-bdc)(2-) = 5-hydroxybenzene-1,3-dicarboxylate and L is a dimethyl sulfoxide, methanol, or water ligand] hydroxylated nanoballs (OH-nanoball) were examined in methanol using optical absorption and steady-state and time-resolved fluorescence methods. The optical spectrum of the OH-nanoball is dominated by ligand absorbance at 305 nm and a weaker Cu(2+)-to-ligand charge-transfer transition at approximately 695 nm, which are distinct from the absorption of either the free ligand (approximately 312 nm) or Cu(2+)(NO(3))(2) (>750 nm) in methanol. The corresponding emission spectrum of the OH-nanoball originates from the emission of the ligand and is centered at approximately 360 nm with a shoulder at approximately 390 nm. The emission from the OH-nanoball is significantly quenched relative to the free ligand [Phi(5-OH-H(2)bdc) = 0.014 and Phi(OH-nanoball) = (5.6 +/- 0.5) x 10-5]. The addition of bases such as imidazole results in an increase in the emission intensity of the OH-nanoball solution, indicating dissociation of the [Cu(2)(5-OH-bdc)(2)L(2)](12) units. Although the mechanism of (5-OH-bdc)(2-) quenching within the OH-nanoball is not clear, it is likely due to interactions between the ligand pi system and the Cu d orbitals. Fluorescence polarization studies further suggest that the OH-nanoball retains a spherical shape in solution. This is evident by the fact that the fluorescence anisotropy of the nanoball is nearly identical with that of the free ligand, suggesting rapid energy transfer (homogeneous fluorescence resonance energy transfer) between ligands within the OH-nanoball.

Binding of bovine prion protein to heparin: A fluorescence polarization study

Glycosaminoglycans (GAGs) are believed to be associated with prion disease pathology and also with metabolism of the prion protein. Fluorescence polarization assay (FPA) of binding between bovine recombinant prion protein (brecPrP) and heparin labelled with AlexaFluor488 was used in model experiments to study glycosaminoglycan–prion protein interaction. Heparin binding to brecPrP was a rapid reversible event which occurred under defined conditions. The interaction of brecPrP with fluorophore-labelled heparin was inhibited by the presence of Cu 2+ ions and was sensitive to competition with heparin, heparan sulphate, and dextran. The dissociation constant of the heparin–brecPrP complex was 73.4 ± 3.7 nM. Circular dichroism (CD) experiments indicated that the structure of brecPrP was less helical in the presence of heparin.

Arsenic Disruption of Steroid Receptor Gene Activation: Complex Dose−Response Effects Are Shared by Several Steroid Receptors

Chronic intake of arsenic (As) has been associated with increased risk of cancer, diabetes, developmental and reproductive problems, and cardiovascular disease. Recent studies suggest increased health risks with drinking water levels as low as 5-10 ppb. We previously reported that As disrupts glucocorticoid receptor (GR) mediated transcription in a very complex fashion. Low As levels (0.1-0.7 microM) stimulated transcription, whereas slightly higher levels (1-3 microM) were inhibitory. The DNA binding domain (DBD) was the minimal region of GR required for the response to As. Mutations in the DBD that alter the conformation of the dimerization domain (D-loop) to a DNA-bound GR conformation abolished the stimulatory effect and enhanced the inhibitory response to As. Here we report that receptors for progesterone (PR) and mineralocorticoids display a complex As response similar to that of the GR, suggesting a common mechanism for this effect. The complex response to As is not due to altered steroid or receptor levels. Moreover, a well-characterized GR dimerization mutant displayed a wild-type biphasic response to As for several divergent reporter genes, suggesting that dimerization is not critical for the response to As. Fluorescence polarization studies with purified PR and GR demonstrated that the specific PR/GR-DNA interaction is not altered in the presence of As. These results indicate that the numerous and diverse human health effects associated with As exposure may be mediated, at least in part, through its ability to simultaneously disrupt multiple hormone receptor systems.

Effect of temperature on aggregation behavior of aqueous solutions of sodium cumene sulfonate

Aqueous solutions of sodium cumene sulfonate are characterized by various experimental techniques for its aggregation behavior as a function of temperature. Surface tensiometry, volumetric changes, vapor pressure osmometry, fluorescence polarization studies and enthalpy changes show changes in the solutions with increasing concentration and temperature. The apparent and partial molal volumes of the hydrotrope, derived from the density measurements, show a sharp increase at a characteristic minimum hydrotrope concentration. The osmotic coefficient of NaCS, obtained from vapor pressure osmometry, shows a significant deviation indicating aggregation of the hydrotrope molecules in aqueous solutions. NaCS, when used as an intrinsic fluorescent probe, reveals a non-polar environment in the aggregate with substantial microviscosity. The enthalpies of micellization obtained directly from the calorimetric measurements show a trend towards lower values with increasing temperature.

Effects of chlorpromazine on plasma membrane permeability and fluidity in the rat brain: A dynamic positron autoradiography and fluorescence polarization study

Antipsychotic drugs have been widely used in psychiatry for the treatment of various mental disorders, but the underlying biochemical mechanisms of their actions still remain unclear. Although phenothiazine antipsychotic drugs have been reported to directly interact with the peripheral plasma membrane, it is not known whether these drugs actually affect plasma membrane integrity in the central nervous system. To clarify these issues, we investigated the effect of chlorpromazine (CPZ), a typical phenothiazine antipsychotic drug, on plasma membrane permeability in fresh rat brain slices using a dynamic positron autoradiography technique and [ 18F]2-fluoro-2-deoxy- d-glucose ([ 18F]FDG) as a tracer. Treatment with CPZ (≥ 100 μM) resulted in the leakage of [ 18F]FDG-6-phosphate, but not [ 18F]FDG, suggesting that the [ 18F]FDG-6-phosphate efflux was not mediated by glucose transporters, but rather by plasma membrane permeabilization. The leakage of [ 18F]FDG-6-phosphate was followed by slower leakage of cytoplasmic lactate dehydrogenase, suggesting that CPZ could initially induce small membrane holes that enlarged with time. Furthermore, the addition of CPZ (≥ 100 μM) caused a decrease in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, which implies an increase in membrane fluidity. CPZ loading dose-dependently increased both membrane permeability and membrane fluidity, which suggested the involvement of a perturbation of membrane order in the mechanisms of membrane destabilization induced by antipsychotic drugs.

Revealing Two-State Protein−Protein Interactions of Calmodulin by Single-Molecule Spectroscopy

We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

Study of the Interaction between Different Phosphodiesterases and Yessotoxin Using a Resonant Mirror Biosensor

Yessotoxins (YTXs) are disulfated polyether toxins that were first isolated from scallops in Japan. It has been proposed that these toxins activate cellular phosphodiesterases (PDEs). The interaction between YTX and PDEs was confirmed by resonant biosensor and fluorescence polarization studies. The aim of this work is to study the specificity of different PDEs for YTX binding. Association measurements are done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers from the sensor surface where the association PDEs-YTX takes place. We use aminosilane cuvettes, where exonuclease Phosphodiesterase I from Crotalus atrox (PDE I), exonuclease Phosphodiesterase II from bovine spleen (PDE II), or phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) are immobilized. Over immobilized exonuclease PDE I and exonuclease PDE II are added different amounts of YTX, and typical association curve profiles are observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration, the association rate constant (k(ass)) and the dissociation rate constant (k(diss)) are obtained. From these values, the kinetic equilibrium dissociation constant (K(D)) of the YTX-PDE association can be calculated, indicating the affinity between them. The specificity of cyclic nucleotide PDE families is studied using different inhibitors that are added over immobilized cyclic nucleotide PDEs. In these conditions, changes in the association PDEs-YTX curves are detected. The results show YTX affinity by cyclic nucleotide PDE 1, PDE 3, PDE 4, and exonuclease PDE I.

Fluorescence polarization and rheological studies of the poly( N-vinyl-2-pyrrolidone) hydrogels produced by UV radiation

Poly( N-vinyl-2-pyrrolidone) hydrogels produced by direct ultraviolet irradiation of PVP aqueous solution leads to crosslinking through pyrrolidinone moiety photolysis. Generally, hydrogel physical properties, like crosslinking density, pore size, swelling capacity, storage and loss moduli are obtained by swelling and rheological tests. However, relations between anisotropy obtained by fluorescence polarization studies and these properties have not been addressed for hydrogel systems. In this work we show that there is a correlation between the data obtained from anisotropy and rheological experiments, since both of them are related with crosslinking density of the hydrogels. These results reveal that fluorescence polarization spectroscopy is a promising tool for understanding the structure of hydrogels.

Characterization of the IKK Signaling Complex

The transcription factor NF-κB is critical to the coordination of both innate and adaptive immune responses. The IκB kinase (IKK) complex is an important regulator of NF-κB activity. Targets that affect NF-κB signaling represent attractive therapeutic strategies for autoimmune diseases. Understanding assembly of the IKK complex is a necessary step in elucidating the molecular function of this complex and provides insight on methods of potential therapeutic intervention. The IKK complex is composed of three subunits: IKKα, IKKβ and NEMO (IKKγ). In this paper analytical ultracentrifugation is used to characterize the homo-oligomerization of the IKK components: α, β and γ as well as the hetero-oligiomerization of the components. The program MULTI-COIL was used to identify a C-terminal region of IKKβ which immediately precedes the NBD (NEMO binding domain) with a propensity to form a coiled-coil. The corresponding sequence from IKKα has a lower propensity. A fluorescent chimeric peptide was made which contains the predicted IKKβ coiled-coil and the IKKα NBD. This peptide binds to a construct containing the first 200 residues of NEMO with a Kd of 90 nM. Fluorescence polarization studies show that IKKβ binds to NEMO with a similar affinity. The affinities of IKKα as well as a synthetic NBD peptide are much lower. We conclude that a coiled-coil interaction in conjunction with the NBD contribute to IKK-NEMO binding.

Dual fluorescence and laser emissions from fluorescein-Na and eosin-B

Dual laser emissions were observed from fluorescein-Na and eosin-B in ethanolic solutions individually in the concentration range from 10 −2 to 10 −3 mol dm −3 under N 2 laser excitation. The first compound was found to lase at two distinct regions with wavelength maxima around 540, 550 nm, while the second one around 558, 574 nm. Steady-state absorption, fluorescence excitation, fluorescence polarization, fluorescence emission and decays of the dyes in various solvents under varying conditions of excitation and detection systems were carried out to identify the nature of the emitting species responsible for laser emissions in two distinct regions. Both the dyes exhibited concentration and excitation wavelength dependence of fluorescence and the effects were found to be more pronounced in binary solution. The fluorescence decays of dyes were monoexponential in ethanol, while in some other solvents used, the decays showed biexponential behavior. The absorption and excitation studies using thin layers of solutions revealed the formation of dimers with the dye concentration around 1×10 −3 mol dm −3. Fluorescence polarization and decay studies confirmed the presence of dimers. The two laser bands observed in the shorter and longer wavelengths were respectively ascribed to monomeric and dimeric species.