POLARIZED PHOTOACOUSTIC SPECTRA OF PHYCOERYTHRIN and PHYCOCYANIN IN ANISOTROPIC POLYMER FILMS

Abstract— Photoacoustic spectra (PAS) of biliproteins, namely, R-phycoerythrin (PE) and C-phycocyanin (PC) and their mixture in anisotropic and isotropic polyvinylalcohol (PVA) films were measured under the illumination with polarized and natural light. Also samples in deuterated PVA were investigated. The yields of fluorescence of various chromophores of investigated biliproteins were obtained from PAS, absorption, fluorescence and fluorescence lifetime measurements. The deuteration of samples causes different changes in thermal deactivation of excitation of various chromophores. Ratios of PAS to absorption of the light polarized parallel and perpendicular to the direction of film stretching are different. The PAS amplitude of deuterated samples is higher than that for undeuterated.     

PHYCOERYTHROCYANIN: ITS SPECTROSCOPIC BEHAVIOR AND PROPERTIES

Abstract— Phycoerythrocyanin is a biliprotein found in very few blue-green algae. Its properties have been examined under three conditions: in whole cells, in light-harvesting organelles (the phycobilisomes). and as an isolated protein. Absorption and fluorescence bands characteristic of the isolated protein are essentially the same as those in intact cells of the blue-green alga Anabaena variabilis. The same spectroscopic hallmarks are observed in purified phycobilisomes. Dissociation of these physobilisomes at low-phosphate concentrations resulted in increased phycoerythrocyanin fluorescence. This time-dependent increase in fluorescence demonstrates the function of this biliprotein in excitation-energy transfer to the other biliproteins when the organelles are intact. The relative stabilities of the various heteroprotein bonds within the phycobilisomes are shown to possess differing phosphate ion dependencies. Studies on the isolated protein from Mastigocladus laminosus include fluorescence measurements at both 23 and-196°C, as is generally observed with biliproteins, although phycoerythrocyanin has complex visible absorption and excitation spectra, only a single emission band is observed.     

POLARIZED PHOTO ACOUSTIC SPECTRA OF PHYCOBILISOMES IN POLYVINYL ALCOHOL FILMS

Abstract— Phycobilisomes from Porphyridium cruentum , suspended in polyvinyl alcohol were found to be highly stable, and had normal absorption and fluorescence spectra. Intact phycobilisomes had a major emission peak at 680 nm, whereas upon partial dissociating the major emission was at 580 nm. Incorporation of phycobilisomes in thin polyvinyl alcohol films facilitated examination by photoacoustic spectroscopy. The photoacoustic spectra had a broad absorption maximum at 545–565 nm (phycoerythrin), which resolved as two peaks (545 and 563 nm) in absorption spectra. Stretching of films resulted in apparent chromophore reorientation in partially dissociated, but not in intact phycobilisomes. Only in dissociated phycobilisomes was observed a differential chromophore orientation at 685 nm by polarized fluorescence, which is attributed to a change in orientation of the terminal phycobilisome pigment relative to phycoerythrin.     

PHOTO ACOUSTIC SPECTRA OF PHYCOBILIPROTEINS and CHLOROPHYLLIN IN ISOTROPIC and ANISOTROPIC POLYVINYL ALCOHOL FILMS

Abstract— Photoacoustic spectra (PAS) of biliproteins: R-phycoerythrin (PE) with R-phycocyanin (PC) admixture in isotropic and anisotropic polyvinyl-alcohol (PVA) films were measured. The mixtures of biliproteins and chlorophyllin (Chllin) in PVA were also investigated. In order to establish the influence of PVA anisotropy on PAS amplitude, the stretched and unstretched PVA films colored by Congo red were studied. The films with PE and Chllin mixture when illuminated in the region of PE absorption, show higher PAS signals than the films with PE alone. This is explained by the excitation energy transfer from efficiently fluorescent PE to Chllin that has much lower yield of fluorescence. The stretched films always show a higher PAS signal than isotropic films because of the increase in the thermal conductivity of PVA. But normalized PAS of Congo red and Chllin in stretched PVA are similar to those in unstretched films.
Pigment molecules oriented parallel to the direction of film stretching have a lower rate of thermal deactivation compared to that of unoriented molecules as has been found by comparing PAS of differently oriented Congo red molecules using photoselection by polarized light. The same result was obtained for various PE chromophores which are differently oriented in anisotropic films.     

PHOTOPHYSICAL PROPERTIES OF PHYCOBILIPROTEINS FROM PHYCOBILISOMES: FLUORESCENCE LIFETIMES, QUANTUM YIELDS, AND POLARIZATION SPECTRA

Abstract— Absorption and fluorescence polarization spectra, as well as absolute fluorescence quantum yields, and lifetimes of phycobiliproteins separated from intact phycobilisomes of Porphyridium cruentum, Nostoc sp. and Fremyella diplosiphon were measured. Two different types of phycoerythrin, in addition to phycocyanin and allophycocyanin, were separated from both Porphyridium cruentum and Nostoc sp. phycobilisomes. They were distinguishable by the shape of their absorption spectra, values of fluorescence quantum yields and their limiting polarization. Phycobilisomes of Fremyella diplosiphon had a type of phycoerythrin that was different from the above kinds. By the use of fluorescence quantum yields and lifetime data, the values of natural lifetimes, the decadic molar extinction coefficients, as well as Förster's critical distances R ₀ for excitation energy transfer, between phycobiliproteins in phycobilisomes, were estimated. The values obtained of Förster's critical distances indicate that for most efficient energy transfer from phycoerythrin to allophycocyanin, the outer layers of Porphyridium cruentum and Nostoc sp. phycobilisomes should be composed of bangiophycean, phycoerythrin and cyanophytan phycoerythrin-II respectively.     

Fluorescence polarization studies of B-phycoerythrin oriented in polymer film

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.     

DELAYED MICROSECOND LUMINESCENCE OF PHYCOBILISOMES

The time-resolved luminescence spectra (in the microsecond range) of phycobilisomes and biliproteins in buffer and polymer matrix were measured in the temperature range from 8 K. to 293 K. Delayed luminescence located in the same spectral region as prompt fluorescence of investigated samples (DLF) and the long-wavelength delayed emission in the720–760 nm range (DL1) was observed. The temperature and viscosity dependencies of DLF and DL1 luminescences were different, but both do not have uniexponential decays and are not quenched by oxygen. This means that delayed luminescence could be generated without the participation of the triplet states, or the chromophores could be shielded by protein against interaction with oxygen. The linear dependence of delayed luminescence on exciting light intensity shows that delayed luminescence is monophotonically induced. It seems that both DLF and DL1 are related to electron-cation recombination, which yields excited singlet states. The DLF is emitted from the first excited singlet state of biliprotein chromophores and DL1 from the same state of the excimers or from the triplet state of some groups of chromophores. Ionization energy of chromophores can be lowered as a result of their interactions with the environment. Delay of emission is due to the trapping or solvation of electrons. Every type of biliprotein consisting of phycobilisomes possesses its own “trap” and can emit the DL. In the case of native phycobilisomes a competition between the excitation energy trapping and transfer occurs.     

EXCITATION ENERGY MIGRATION IN PHYCOBILISOMES: COMPARISON OF EXPERIMENTAL RESULTS AND THEORETICAL PREDICTIONS

Abstract— Quantum yield and fluorescence polarization determinations on phycobilisomes and their constituent phycobiliproteins show that phycobilisomes are energetically effective macromolecular structures. Energy migration within the phycobilisome to allophycocyanin, the longest wavelength absorbing and emitting phycobiliprotein, was indicated by the predominant allophycocyanin fluorescence emission which was independent of the phycobiliprotein being excited. The high efficiency of the energy migration inside the phycobilisome was reflected by the low polarized fluorescence. Excitation of phycobilisomes in the region of major absorption (500–650 nm) resulted in degrees of fluorescence polarization between +0.02 and –0.02, whereas in isolated phycobiliproteins the values were 2 to 12 times greater. Furthermore, 94–98° of the excitation energy of phycoerythrin was transferred to phycocyanin and allophycocyanin as determined from comparisons of fluorescence spectra of intact and dissociated phycobilisomes. The fluorescence quantum yields of phycobilisomes were about 0.60–0.68, very similar to that of pure allophycocyanin in solution (0.68). Phycobilisomes isolated from Fremyella diplosiphon and Nostoc sp. (blue-gree algae) have respective quantum yields of 0.68 and 0. 65, and those isolated from Porphyridium cruentum (red alga), about 0.60. In Fremyella diplosiphon and Nostoc sp., which showed a striking adaptation to different wavelengths, the phycobilisome quantum yields only varied from 0.68 to 0.67 and from 0.65 to 0. 60, respectively. The mean transfer time, calculated on the basis of experimental results, was about 280 ± 40 ps for transfer of excitation from the phycoerythrin to the phycocyanin layer in phycobilisomes. This time corresponds to the mean number of jumps, about 28, of the excitation in the phycoerythrin layer before it is captured by phycocyanin. These values are in reasonable agreement with the values of 250 ± 30 ps and 25 jumps, calculated on the basis of a phycobilisome model (of Porphyridium cruentum) and Pearlstein's theory of energy migration devised for a three-dimensional photosynthetic unit. It was also shown that Paillotin's theory of energy migration predicts similar values for mean transfer time and mean number of jumps, if one assumes that phycocyanin is a perfect sink for phycoerythrin excitation.     

PHYCOBILISOMES AND ISOLATED PHYCOBILIPROTEINS. EFFECT OF GLUTARDIALDEHYDE AND BENZOQUINONE ON FLUORESCENCE

Abstract— The fluorescence of the biliproteins C-phycocyanin from Spirulina platensis , B-phycoerythrin from Porphyridium cruentum and of isolated whole P. cruentum phycobilisomes is quenched in the presence of glutardialdehyde (GA) or benzoquinone (BQ). The kinetics of fluorescence decrease thus induced is biphasic. If GA is used as a quencher, the fluorescence can be recovered at 77 K. Contrary to the GA-effect, only a minor recovery takes place with BQ at 77 K, thus demonstrating a different mechanism of action of GA and BQ on biliproteins.     

多变鱼腥藻藻胆蛋白共价复合物的合成及其分子内能量传递

利用双功能基偶联剂3-(2-吡啶联巯基)丙酸N-羟基琥珀酰亚胺酯(SPDP)合成了两个藻胆蛋白复合物,藻红蓝蛋白-变藻蓝蛋白复合物PEC-APC和藻红蓝蛋白-藻蓝蛋白复合物PEC-PC.利用吸收光谱和荧光光谱证明了藻胆蛋白构型与构象在反应后得到保持。通过荧光光谱观察到能量传递现象。计算出复合物PEC-APC的分子内能量传递效率约为90%.复合物PEC-PC中藻红蓝蛋白PEC的荧光寿命比PEC本身的寿命大大缩短,证明存在分子内能量传递。二硫苏糖醇(DTT)还原二硫桥键后能量传递被阻断。这进一步证明复合物合成成功及分子内能量传递。     

Site, rate, and mechanism of photoprotective quenching in cyanobacteria

In cyanobacteria, activation of the Orange Carotenoid Protein (OCP) by intense blue-green light triggers photoprotective thermal dissipation of excess absorbed energy leading to a decrease (quenching) of fluorescence of the light harvesting phycobilisomes and, concomitantly, of the energy arriving to the reaction centers. Using spectrally resolved picosecond fluorescence, we have studied cells of wild-type Synechocystis sp. PCC 6803 and of mutants without and with extra OCP (ΔOCP and OverOCP) both in the unquenched and quenched state. With the use of target analysis, we managed to spectrally resolve seven different pigment pools in the phycobilisomes and photosystems I and II, and to determine the rates of excitation energy transfer between them. In addition, the fraction of quenched phycobilisomes and the rates of charge separation and quenching were resolved. Under our illumination conditions, ～72% of the phycobilisomes in OverOCP appeared to be substantially quenched. For wild-type cells, this number was only ～29%. It is revealed that upon OCP activation, a bilin chromophore in the core of the phycobilisome, here called APC(Q)(660), with fluorescence maximum at 660 nm becomes an effective quencher that prevents more than 80% of the excitations in the phycobilisome to reach Photosystems I and II. The quenching rate of its excited state is extremely fast, that is, at least (～240 ± 60 fs)(-1). It is concluded that the quenching is most likely caused by charge transfer between APC(Q)(660) and the OCP carotenoid hECN in its activated form.     

POLARIZED SPECTRA OF ORIENTED PIGMENT-LIPOPROTEIN COMPLEXES FROM THE PURPLE BACTERIUM: Chromatium minutissimum

Abstract— Polarized absorption, fluorescence and photoacoustic spectra of bacteriochlorophyll (BChl)-lipoprotein complexes from the purple bacterium Chromatium minutissimum oriented in stretched polyvinylalcohol films were measured at room temperature and 85 K. The preparations contain large amounts of the B800-820 antenna complexes. From polarized absorption spectra taken under various light beam incidence angles with respect to the film plane, conclusions concerning arrangement of pigment molecules in B800-820 complex are obtained. The transition moments of the BChl Q_y band are not exactly parallel to the membrane plane. It seems that there are pools of differently oriented BChl chromophores absorbing in both 800 nm and 820 nm regions. Change in temperature strongly influences linear dichroism of carotenoids and BChl Q_y bands. The reversible changes in absorption, linear dichroism and photoacoustic spectra caused by the variation in sample temperature suggest strongly the reversible twisting of carotenoid molecules, related probably to modification of the interactions between carotenoids and proteins. Various carotenoids exhibit different yield of thermal deactivation and this yield is also temperature dependent.     

Optical Spectra of Polygermane/Mesoporous Silica Nanocomposites

Summary: The monitoring of poly(di-n-hexylgermane) (PDHG) optical spectra in a variety of structures ranging from a bulk film to a nanosize polymer confined into a nanopore of SBA-15 was performed using the fluorescence and fluorescence excitation spectra in the temperature range from 5 to 240 K as well as the absorption and FTIR spectra at room temperature. The observed data were compared with those obtained for poly(di-n-hexylsilane). It was shown that PDHG film absorption and fluorescence spectra strongly depend on the polymer thickness and consist of a number of bands which were assigned to centers with different amount of trans- and gauche- conformers of the polymer chains. Conformations of the polymer chains found in a thin film and in a 10 nm pore are similar while differing from the conformations of a thick film. Optical spectra of the confined PDHG are blue-shifted relative to those of a thin film. The PDHG polymer chain conformation becomes disordered with the decrease of the polymer film thickness and the nanopore size from 10 to 6 nm.     

INCREASED ENERGY TRANSFER FROM PHYCOBILISOMES TO PHOTOSYSTEM II IN HIGH LIGHT ADAPTED Anabaena cylindrica

Excitation energy transfer from phycobilisomes to photosystem II in high-light adapted cells of Anabaena cylindrica was studied by fluorescence spectroscopy and compared to that of low-light adapted cells. Measurements were made on membrane fragments containing phycobilisomes, photosystem I and II, isolated in 0.75 M K-phosphate. Relative efficiency of 430 to 590 nm light in the excitation of F₆₈₀ chlorophyll fluorescence was compared in low and high light adapted cells, respectively. The values indicate that light energy absorbed by phycobilisomes is transferred to photosystem II antenna chlorophylls with higher efficiency in high-light adapted cells than in low-light adapted cells. Partial dissociation and uncoupling of energy transfer caused by low ion concentration were different in the membrane fragments isolated from the two kinds of cells and indicated a higher aggregation state of pigment-protein complexes of phycobilisomes in high-light adapted A. cylindrica cells.     

POLARIZED TIME-RESOLVED FLUORESCENCE SPECTRA OF PHYCOBILISOMES ISOLATED FROM Tolypothrix tenuis EMBEDDED IN POLY(VINYL ALCOHOL) FILM

–Time-resolved fluorescence spectra in the ps time range were measured on phycobilisome (PBS) embedded in poly(vinyl alcohol) films. The cyanobacterium Tolypothrix tenuis was used as a source of PBS because the pigment composition and the structure of the PBS are well defined. Isotropic PBS in the unstretched film and PBS uniaxially oriented in the stretched film were investigated. Diameters of PBS hemidiscs were oriented parallel to the film-stretching direction. The time-resolved fluorescence spectra of the unstretched sample and of the two polarized components in the stretched samples showed several differences in the rise and decay. The delay time, estimated from the time span between the maximum laser pulse and maximal intensity of the phycocyanin and allophycocyanin fluorescence, was much longer than that reported in the aqueous media. This suggests occurrence of a higher thermal deactivation of PBS in polymer film than in aqueous media. The excitation energy transfer from excited phycocrythrin to allophycocyanin was more efficient in the unstretched than in the stretched samples, and it was greater in the parallel polarized component of the stretched sample than in the perpendicular component. The present results are in agreement with a previous suggestion which states that there are two independent pathways of excitation energy transfer in PBS and that there is more than one final emitter of fluorescence. The molecules taking part in various pathways of energy transfer differ in their orientation within PBS.     

ENERGY TRANSFER STUDIES ON CRYPTOMONAD BILIPROTEINS

Abstract. Fluorescence techniques of various types have been used to study the light-gathering and energy transfer modes for various cryptomonad biliproteins (phycocyanin or phycoerythrins). Analysis of fluorescence polarization and absorption data demonstrates that each cryptomonad biliprotein is composed of at least two distinct types of absorbing chromophore, each attached to the protein through covalent linkages to different polypeptide chains. Examination of the fluorescence emission spectra as a function of excitation at several wavelengths demonstrates that only one of these absorbing chromo-phores is responsible for the fluorescence. This behavior is consistent with a known phenomenon whereby photons are gathered by more than one chromophore and then after radiationless energy transfer are emitted by only one chromophore. Application of Förster dipole-dipole energy transfer theory is made to the study of the mode by which energy absorbed by biliproteins migrates to Chl a. The spectral overlap integral between phycocyanin (Chroomonas sp.) and Chl a is 7.13 ± 10^-10cm⁶mol^-1and between phycocyanin and Chl c₂ 0.25 ± 10^-10cm⁶mol^-1. This large difference in overlap suggests, although does not prove, that phycocyanin might transfer energy directly to Chl a without a Chl c₂ intermediary. The cryptomonad phycoerythrins may also use this method but a Chl c₂ intermediate could not be ruled out for them. Radiationless energy transfer among homogeneous biliproteins is shown to be feasible. All these calculations are based on in vitro spectra and the interpretations extrapolated to the cellular situation, and these tentative conclusions are reached without knowledge of other factors, such as chromophore-chro-mophore orientation and distance, which could greatly influence the energy transfer scheme.     

ENERGY STORAGE IN THE PRIMARY PHOTOREACTION OF BOVINE RHODOPSIN. A PHOTOACOUSTIC STUDY

Abstract— Photoacoustic spectroscopy is used to investigate the uptake of energy in the primary photoreaction of bovine visual pigment (Rhodopsin → Bathorhodopsin). It is shown that very concentrated dried rod outer segment membranes have a sufficient thermal diffusivity to be analyzed by this technique. From the photoacoustic and absorption spectra of these membranes, the low temperature dissipation spectrum has been obtained and the results are consistent with the storage of 145 kJ mol^-1 in the primary event of vision having a quantum yield of 0.67. By photoacoustic spectroscopy, this process is continuously monitored from 350 to 550 nm and its efficiency is found to vary by less than 10%, even in the spectral region of the β-band of rhodopsin.     

Photophysical processes in aromatic polyimides. Studies with model compounds

Emission mechanism in an aromatic polyimide, PI(BPDA/PDA), derived from biphenyltetracarboxylic dianhydride and p-phenylene diamine were studied with ultraviolet visible absorption and fluorescence spectra of a series of the model compounds. The excitation spectrum of the intermolecular charge-transfer (CT) fluorescence peaking around 550 nm of PI(BPDA/PDA) thin film was completely consistent with the absorption spectrum, indicating that the intermolecular CT fluorescence emission of PI(BPDA/PDA) film is not caused by direct excitation of the CT absorption band, but by light absorption due to structural units in the polymer backbone. The UV-vis. absorption spectra of the model compounds corresponding to the structural units in PI(BPDA/PDA) showed that the longest wavelength absorption band is due to the biphenylbisimide moiety. The band was assigned as π, π* transition with the polarization spectrum of the model compound. The fluorescence spectra of the model compounds changed sensitively depending on the conformation around N-phenyl bond. The lifetime measurement for the model compounds suggested that intramolecular CT process occurs very rapidly. © 1993 John Wiley & Sons, Inc.     

Influence of intramolecular hydrogen bonding on OH-stretching overtone intensities and band positions in peroxyacetic acid

Vapor phase absorption spectra and integrated band intensities of the OH stretching fundamental as well as first and second overtones (2ν(OH) and 3ν(OH)) in peroxyacetic acid (PAA) have been measured using a combination of FT-IR and photoacoustic spectroscopy. In addition, ab initio calculations have been carried out to examine the low energy stable conformers of the molecule. Spectral assignment of the primary features appearing in the region of the 2ν(OH) and 3ν(OH) overtone bands are made with the aid of isotopic substitution and anharmonic vibrational frequency calculations carried out at the MP2/aug-cc-pVDZ level. Apart from features associated with the zeroth-order OH stretch, the overtone spectra are dominated by features assigned to combination bands composed of the respective OH stretching overtone and vibrations involving the collective motion of several atoms in the molecule resulting from excitation of the internal hydrogen bonding coordinate. Integrated absorption cross section measurements reveal that internal hydrogen bonding, the strength of which is estimated to be ～20 kJ/mol in PAA, does not result in a enhanced oscillator strength for the OH stretching fundamental of the molecule, as is often expected for hydrogen bonded systems, but does cause a precipitous drop in the oscillator strength of its 2ν(OH) and 3ν(OH) overtone bands, reducing them, respectively, by a factor of 165 and 7020 relative to the OH stretching fundamental.     

Relaxation of photo-excitations in films of oligo- and poly-(para-phenylene vinylene) derivatives

The photo-luminescence from solid films of poly(para-phenylene vinylene) polymers and an oligomeric model system, consisting of seven repeat units, are investigated at low temperature (8 K) using time-resolved spectroscopic techniques. Results are compared to those for the materials in solution. In the case of the oligomer, the shape of the visible absorption band observed for the film is quite different from the band shape for the polymer in frozen solution and is characteristic of H-type aggregates. Theoretical models are presented describing the dependence of the band shape of absorption and emission spectra on intermolecular excited state interactions, electron-vibration coupling and disorder represented by distributions of the molecular excitation and intermolecular interaction energies. Using these models, it is concluded that intermolecular interactions in the film of the oligomer are strong (1400 cm⁻¹), and the disorder low, implying delocalization of the excitation over several molecules. In accordance with these models the fluorescence lifetime for the film (2 ns) is considerably longer than for isolated molecules in solution (0.45 ns). The emission spectra of the film, taken early after excitation, are consistent with delocalization of the excitation over several molecules. A time-dependent red shift of the fluorescence band is observed and interpreted in terms of migration of localized excitations between disorder induced trap sites, which exist in the low energy tail of the density of excited states. For the polymers, differences between the shape of the absorption bands of solid film and frozen solution are smaller than for the oligomer indicating that interchain interactions that are, on average, weaker than for the oligomer. For the polymer films, a time-dependent red shift of the emission is observed and fluorescence depolarization measurements provide direct evidence for migration of the photo-excitations between trap sites. For one polymer, a time dependent change in the band shape of the fluorescence after pulsed excitation is observed with the band shape of the long-lived emission being compatible with that expected for an excitation delocalized over at least two, nearly parallel aligned, chains. For a second polymer, the emission band shape and its time evolution indicate that the major part of the fluorescence originates from disorder induced luminescent sites. These results indicate that the spectroscopic properties of films of π-conjugated polymer critically depend on parameters such as density of defects and excited state interchain interaction energy.