Cyclodextrin insulation prevents static quenching of conjugated polymer fluorescence at the single molecule level.

Conjugated polymers (CPs) are promising materials for fluorescence imaging application. However, a significant problem in this field is the unexplained abnormally low fluorescence brightness (or number of fluorescence photons detected per one excitation photon) exhibited by most of CP single chains in solid polymer hosts. Here it is shown that this detrimental effect can be fully avoided for short chains of polyfluorene-bis-vinylphenylene (PFBV) embedded in a host polymer matrix of PMMA, if the conjugated backbone is insulated by cyclodextrin rings to form a polyrotaxane (PFBV-Rtx). Fluorescence kinetics and quantum yields are measured for the polymers in liquid solutions, pristine films, and solid PMMA blends. The fluorescence brightness of PFBV-Rtx single chains dispersed in a solid PMMA is very close to that expected for a chain with 100% fluorescence quantum yield, while the unprotected PFBV chains of the same length possess 4 times lower brightness. Despite this, the fluorescence decay kinetics are the same for both polymers, suggesting the presence of static or ultrafast fluorescence quenching in the unprotected polymer. About 80% of an unprotected PFBV chain is estimated to be completely quenched. The hypothesis is that the cyclodextrin rings prevent the quenching by working as 'bumpers' reducing the mechanical forces applied by the host polymer to the conjugated backbone and help retaining its conformational freedom. While providing a recipe for making CP fluorescence bright at the single-molecule level, these results identify a lack of fundamental understanding in the community of the influence of the environment on excited states in conjugated materials.

Polarization single complex imaging of circular photosynthetic antenna.

Single complex fluorescence polarization spectroscopy is applied to study the peripheral light harvesting antenna (LH2) from photosynthetic purple bacterium Rhodopseudomonas (Rps.) acidophila. The measured two-dimensional excitation-emission polarization plots are used to construct geometric representation for the absorbing B800 and emitting B850 as ellipses. The shape and orientation of the ellipses is discussed in terms of tilted LH2 complexes where emission occurs from energetically disordered B850 excitons.

Organization of bacteriochlorophylls in individual chlorosomes from Chlorobaculum tepidum studied by 2-dimensional polarization fluorescence microscopy.

Chlorosomes are the largest and most efficient natural light-harvesting systems and contain supramolecular assemblies of bacteriochlorophylls that are organized without proteins. Despite a recent structure determination for chlorosomes from Chlorobaculum tepidum (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525), the issue of a possible large structural disorder is still discussed controversially. We have studied individual chlorosomes prepared under very carefully controlled growth condition by a novel 2-dimensional polarization single molecule imaging technique giving polarization information for both fluorescence excitation and emission simultaneously. Contrary to the existing literature data, the polarization degree or modulation depth (M) for both excitation (absorption) and emission (fluorescence) showed extremely narrow distributions. The fluorescence was always highly polarized with M ≈ 0.77, independent of the excitation wavelength. Moreover, the fluorescence spectra of individual chlorosomes were identical within the error limits. These results lead us to conclude that all chlorosomes possess the same type of internal organization in terms of the arrangement of the bacteriochlorophyll c transition dipole moments and their total excitonic transition dipole possess a cylindrical symmetry in agreement with the previously suggested concentric multitubular chlorophyll aggregate organization (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525).

Correlation analysis of fluorescence intensity and fluorescence anisotropy fluctuations in single-molecule spectroscopy of conjugated polymers.

Single-molecule spectroscopy techniques are used to investigate time fluctuations of the fluorescence properties of two different types of conjugated polymer, a polythiophene derivative (PDOPT) and a phenylene vinylene derivative (MEH-PPV), at 100 and 293 K. Linear correlation coefficients between fluorescence intensity and polarization are used to characterize fluctuations. We are able to distinguish between different blinking and bleaching effects on the polarization. Furthermore, the polarization data reveal clear differences in the topology of these two polymers, which is related to the ordered conformation of the MEH-PPV. Plots of correlation coefficients appear to be very different for the two polymers and are also very sensitive to temperature. These observations prove that correlation analysis is a useful tool to understand fluorescence fluctuations in multi-chromophoric systems.

Single chain versus single aggregate spectroscopy of conjugated polymers. Where is the border?

Single chains of conjugated polymers e.g. MEH-PPV (poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene) have become interesting objects for single molecule spectroscopy (SMS) studies. However, most of the experiments so far were performed without full awareness of the isolation status of the polymer chains in host matrices. We used steady-state and time-resolved fluorescence methods and 2D polarization single molecule imaging technique to unravel the isolation/aggregation status of MEH-PPV in spin-coated films prepared at different conditions. It turned out that a sample showing isolated bright spots in fluorescence images could be obtained in a very broad concentration range of MEH-PPV when toluene was used as a solvent and PMMA as a matrix. If the MEH-PPV concentration was not sufficiently low, a substantial fraction of the fluorescence spots should be assigned to individual nano-aggregates rather than truly isolated chains of the polymer. Contrary to single aggregates, truly isolated MEH-PPV chains showed blue-shifted emission spectra, mono-exponential fluorescence decay dynamics with relatively long lifetimes (0.4-1.2 ns), and high polarization anisotropy. We argue that insufficient control of the concentration in the published SMS studies of MEH-PPV resulted in incorrect assigning of some spectroscopic properties of single aggregates to isolated MEH-PPV chains. We believe this to be the main origin of discrepancies among the published data in this field.

Fate of excitations in conjugated polymers: single-molecule spectroscopy reveals nonemissive "dark" regions in MEH-PPV individual chains.

Single chains of the conjugated polymer MEH-PPV (poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene)) were studied with wide-field fluorescence microscopy (dispersion in inert polymer matrices) and with fluorescence correlation spectroscopy (chloroform solution). The fluorescence yield of individual molecules in matrices was found to be 1-2 orders of magnitude lower than that in solution and it decreased substantially with increasing chain length. It suggests that isolation of MEH-PPV molecules in polymer matrices creates favorable conditions for photogeneration of nonemissive primary excited states.

Oligothiophene assemblies defined by DNA interaction: from single chains to disordered clusters.

The organization of conjugated polyelectrolytes (CPEs) interacting with biomolecules sets conditions for the biodetection of biological processes and identity, through the use of optical emission from the CPE. Herein, a well-defined CPE and its binding to DNA is studied. By using dynamic light scattering and circular dichroism spectroscopy, it is shown that the CPE forms a multimolecule ensemble in aqueous solution that is more than doubled in size when interacting with a small DNA chain, while single chains are evident in ethanol. The related changes in the fluorescence spectra upon polymer aggregation are assigned to oscillator strength redistribution between vibronic transitions in weakly coupled H-aggregates. An enhanced single-molecule spectroscopy technique that allows full control of excitation and emission light polarization is applied to combed and decorated lambdaDNA chains. It is found that the organization of combed CPE-lambdaDNA complexes (when dry on the surface) allows considerable variation of CPE distances and direction relative to the DNA chain. By analysis of the polarization data energy transfer between the polymer chains in individual complexes is confirmed and their sizes estimated.

Polarization portraits of single multichromophoric systems: visualizing conformation and energy transfer.

A novel technique, two-dimensional (2D) polarization single-molecule imaging, is presented. It is based on measurements and analysis of fluorescence intensity as a function of excitation and emission polarization angles. The technique allows recording of full information on the steady-state polarization properties of fluorescent objects. It is particularly suitable for application to single multichromophoric systems (molecules or nanoparticles) with energy transfer (ET) between different chromophores (e.g., single fluorescent pi-conjugated polymer chains). The 2D polarization data simultaneously provide information on the conformation of the system and the efficiency of its internal excitation ET. The technique is used to characterize single chains and different kinds of chain aggregates of different conjugated polymers at different temperatures. The 2D polarization measurements reveal a dramatic difference in ET taking place in these systems. Clear temperature dependence of ET is observed for individual aggregates as well as for their statistical ensembles. Also, a dependence on solvent and aggregate size is shown. Additionally, extensive "traditional one-dimensional" polarization results on the polarization anisotropy of fluorescence excitation and emission are presented. These results and findings are discussed in relation to internal organization of the nano-objects under study.

Fluorescence blinking, exciton dynamics, and energy transfer domains in single conjugated polymer chains.

In order to understand exciton migration and fluorescence intensity fluctuation mechanisms in conjugated polymer single molecules, we studied fluorescence decay dynamics at "on" and "off" fluorescence intensity levels with 20 ps time resolution using MEH-PPV [poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] dispersed in PMMA. Two types of intensity fluctuations were distinguished for single chains of conjugated polymers. Abrupt intensity fluctuations (blinking) were found to be always accompanied by corresponding changes in fluorescence lifetime. On the contrary, during "smooth" intensity fluctuations no lifetime change was observed. Time-resolved data in combination with data on fluorescence emission and excitation anisotropy lead to a picture where a single polymer molecule is seen as consisting of several energy transfer domains. Exciton migration is efficient within a domain and not efficient between domains. Each domain can have several emitting low-energy sites over which the exciton continuously migrates until it decays. Emission of individual domains is often highly polarized. Fluorescence from a domain can be strongly quenched by Forster energy transfer to a quencher (hole polaron) if the domain overlaps with the quenching sphere.

Host matrix dependent fluorescence intensity modulation by an electric field in single conjugated polymer chains.

An electric field oscillating at a frequency approximately 1 Hz is found to induce strong modulation of the fluorescence intensity of single poly[2-methoxy,5-(2'-ethyl-hexyloxy)-p-phenylene vinylene] (MEH-PPV) molecules (MW approximately 10(6)) embedded in a poly(methyl methacrylate) (PMMA) matrix. The MEH-PPV polymer chains are carefully isolated from the electrodes to avoid effects of injection. In a polystyrene matrix, fluorescence intensity modulations are on average much less pronounced. The difference in average modulation depth can be explained in terms of lower field-induced exciton dissociation rates in the MEH-PPV/polystyrene system compared to MEH-PPV/PMMA because of a lack of suitable acceptor sites. The observed electric field dependence of single-molecule fluorescence strongly suggests that energy transfer from singlet or even triplet excitons to long-living on-chain hole polarons contributes to the observed modulations. The observed large qualitative differences between the responses of different molecules probably reflect differences in chain topology and strongly anisotropic distributions of acceptor sites, while the hysteretic response of some molecules indicates conformational switching.

Excitation polarization provides structural resolution of individual non-blinking nano-objects.

We propose to combine the method of fluorescence intensity centroid localization with rotation of the plane of excitation polarization. Polarized light interacts selectively with differently oriented fluorophores; thus yielding topological information on the nanometer scale, without any need for fluorophore blinking. The method is applicable to photostable individual systems, when most of the traditional super-resolution methods fail. A theoretical study is supported by experiments on 30 nm long cyclodextrin-encapsulated single polyrotaxane conjugated polymer chains.

Inhomogeneous Quenching as a Limit of the Correlation Between Fluorescence Polarization and Conformation of Single Molecules.

The photophysical properties of conjugated polymers (CPs) largely depend on the interactions between the CP and its environment. We present a study of two polymers with identical conjugated backbones, bare and insulated, that showed different fluorescence excitation modulation depth histograms. However, the polarization differences are not related to differences in conformation, as commonly believed, but to the existence of "dark" chromophores in the bare polymer that are statically quenched. This results in inhomogeneous quenching of the polymer chain that breaks the correlation between excitation fluorescence polarization and conjugated polymer chain conformation.

Temperature-dependent exciton dynamics in J-aggregates-when disorder plays a role.

Absorption and fluorescence spectral properties of J-aggregates of core-tetrasubstituted perylene bisimide (PBI) dyes have been investigated in the temperature range from 300 to 5 K. A special feature of the present PBI J-aggregates is that, in contrast to classical J-aggregates like that of pseudoisocyanine (PIC), the chromophores are held together not only by van-der-Waals interactions but also by hydrogen bonds. The temperature dependence of the spectral band widths and the Stokes shift for PBI 1 aggregates are compared with those for 3,3'-bis-(3-sulfopropyl)-5,5'-dichloro-9-ethylthiacarbocyanine (THIATS) J-aggregates, which have been studied in detail previously (J. Phys. Chem. B 2001, 105, 4636-4646). The assemblies of PBI 1 possess very broad absorption bands compared to those of THIATS, even at low temperature (5 times broader). We interpret this observation as an indication for a very large level of disorder in the case of PBI 1 assemblies. Possible sources of disorder in aggregates of PBI 1 are discussed. Despite large quantitative differences, both aggregates show qualitatively the same temperature dependence of the Stokes shift calculated in units of fluorescence bandwidth. The observed temperature dependences are discussed in terms of exciton thermal population of different states of disordered aggregates. Unexpectedly low thermal activation energy of exciton migration is observed for PBI 1 aggregates.