Polarized Fluorescence Excitation Research Articles

High-pressure control of photosynthetic excitons

In photosynthesis, collective pigment excitations – excitons – facilitate chemical reactions for sustainable biological function. Here, the effect of hydrostatic pressure – an important thermodynamic stress factor – on optical spectral properties of excitons in the cyclic light-harvesting 2 pigment-protein complex from photosynthetic bacterium Rhodoblastus acidophilus was first studied. The high pressure-induced modifications of absorption, fluorescence and polarized fluorescence excitation spectra were theoretically analyzed in terms of the disordered molecular exciton model. We uniquely show that the observed shift of the spectra under pressure is largely governed by the pressure-induced rise of two factors: the exciton displacement energy and the exciton coupling energy, which increases the spread of the exciton state manifold. A significant increase of static energy disorder revealed by model calculations suggests that high-pressure compressing of the complex is actually accompanied by altered spatial orientations and conformations of the protein-embedded pigment molecules.

Polarized Localization Microscopy Detects Membrane Curvature and Reveals the Interplay between Membrane Orientation and Protein Dynamics

Nanoscale membrane curvature is a necessary component of countless cellular processes. However, the understanding of these processes is limited by the experimental techniques and its inability to detect and resolve nanoscale membrane curvature. Here we present Polarized Localization Microscopy (PLM), a novel optical microscopy technique that detects and resolves membrane curvature with 15 nm resolution. PLM combines the advantages of polarized total internal reflection fluorescence microscopy (TIRF) and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without sacrificing the localization precision of the point spread function. We demonstrate PLM capability of resolving membrane orientations by draping a supported lipid bilayer over polystyrene nanoparticles of varying sizes on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, we introduce theoretical analysis that enables mapping out the membrane topology from the density of detected localizations in fluorescence excitation polarization. Using this approach, the interplay between membrane topology and cholera toxin subunit B (CTxB) dynamics was addressed. CTxB partitioned to curved membrane over the nanoparticle and further drove formation of lipid tubules. PLM detected the binding of an exocytosis protein complex to nanoscale membrane curvature. PLM was utilized to perform high throughput single particle tracking to reveal the correlated effects of membrane curvature, dynamics, and protein distribution. By providing super-resolution of membrane topology, PLM is able to reveal the critical components in various cellular processes such as endocytosis/exocytosis, viral infections, and neurotransmission.

Effect of the Side‐Chain‐Distribution Density on the Single‐Conjugated‐Polymer‐Chain Conformation

The spatial arrangement of the side chains of conjugated polymer backbones has critical effects on the morphology and electronic and photophysical properties of the corresponding bulk films. The effect of the side-chain-distribution density on the conformation at the isolated single-polymer-chain level was investigated with regiorandom (rra-) poly(3-hexylthiophene) (P3HT) and poly(3-hexyl-2,5-thienylene vinylene) (P3HTV). Although pure P3HTV films are known to have low fluorescence quantum efficiencies, we observed a considerable increase in fluorescence intensity by dispersing P3HTV in poly(methyl methacrylate) (PMMA), which enabled a single-molecule spectroscopy investigation. With single-molecule fluorescence excitation polarization spectroscopy, we found that rra-P3HTV single molecules form highly ordered conformations. In contrast, rra-P3HT single molecules, display a wide variety of different conformations from isotropic to highly ordered, were observed. The experimental results are supported by extensive molecular dynamics simulations, which reveal that the reduced side-chain-distribution density, that is, the spaced-out side-chain substitution pattern, in rra-P3HTV favors more ordered conformations compared to rra-P3HT. Our results demonstrate that the distribution of side chains strongly affects the polymer-chain conformation, even at the single-molecule level, an aspect that has important implications when interpreting the macroscopic interchain packing structure exhibited by bulk polymer films.

Quantitative characterization of light-harvesting efficiency in single molecules and nanoparticles by 2D polarization microscopy: Experimental and theoretical challenges

General problem of extracting intramolecular energy transfer information from fluorescence and fluorescence excitation polarization experiments at single molecule level is presented. A single funnel approximation is shown to be a very powerful approach to model the polarization data obtained by recently emerged 2-dimensional polarization single molecule imaging technique [O. Mirzov et al., Small 5 (2009) 1877]. Using this approximation a parameter characterising quantitatively light-harvesting efficiency of an individual light-harvesting antenna can be readily obtained. Technical details of 2D polarization imaging and practical methods of avoiding polarization artefact in fluorescence microscopy are discussed.

Regioregularity and Single Polythiophene Chain Conformation

The regioregularity of a conjugated polymer can greatly affect bulk film morphologies and properties. However, it remains unclear how regioregularity affects the conformation of isolated individual chains where interchain interactions are absent. Here, the effect of the regioregularity on the conformations adopted by regioregular (rr-) and regiorandom (rra-) poly(3-hexylthiophene) (P3HT) chains was studied using single molecule fluorescence excitation polarization spectroscopy. While every rr-P3HT chain within an ensemble was found to fold into a highly ordered conformation, single rra-P3HT chains adopted a wide variety of conformations, ranging from highly ordered to isotropic. This distribution is likely due to variations in the positions of nonhead-to-tail linkages of the side-chains along the backbone of the different polymer chains. Molecular dynamics simulation on atomistic models of rr- and rra-P3HT chains supports the effect of regioregularity on the collapsed conformations. These results demonstr...

Spectral fine-tuning in excitonically coupled cyclic photosynthetic antennas

Photosynthetic organisms are famously adaptable to environmental variances, yet the underlying physical mechanisms are seldom known. Here, we study the mechanisms that are responsible for the very large (∼50-nm) blue shift of the lowest-energy absorption band in the stressed LH3 antenna form from the photosynthetic bacterium Rhodopseudomonas acidophila compared to its position in regular LH2 antenna. By analyses of the antenna absorption and polarized fluorescence excitation spectra measured at 5 K, essential modifications of the antenna excitons have been revealed that involve both relative narrowing and shifting of the exciton band.

Electronic absorption and emission spectra of Alq 3 in solution with special attention to a delayed fluorescence

Tris(8-quinolinolato)aluminum(III) (Alq 3) shows electronic absorption bands at 378, 360 (in a 1:1 mixed solvent of methanol and ethanol (ME) at 77 K), 334, 316, 300, 263, 255.8, and 233 nm in ethanol at room temperature. According to the polarized fluorescence excitation spectrum together with MO calculations, for instance, the 360 nm band is assigned to an LL CT transition (an intramolecular charge transfer transition between two ligands), and the 378 nm band to an LM/ML CT one (an intramolecular charge transfer transition between ligand and metal). Alq 3 shows a broad fluorescence band peaking at around 478 nm in the ME matrix at 77 K. The emission spectrum measured with a phosphoroscope has two emission bands at 567 and 478 nm. The 567 nm band accompanies vibronic bands at 578 and 605 nm, being safely assigned to a phosphorescence of Alq 3. The lifetimes of the 478 and 567 nm bands are both 5.4 ms. The lifetime of the 478 nm band together with the band position and its band shape indicate that this band can be assigned to a delayed fluorescence.

A disordered polaron model for polarized fluorescence excitation spectra of LH1 and LH2 bacteriochlorophyll antenna aggregates

Excitonic polarons in antenna complexes are subject to static lattice disorder. A model has been developed to analyze polarized fluorescence excitation spectra of circular light-harvesting complexes from purple photosynthetic bacteria containing bacteriochlorophyll as the main photoactive pigment that includes both diagonal (energetic) and off-diagonal (structural) disorders. Essential differences of disorder realizations seem to exist between the core LH1 and peripheral LH2 complexes from the bacterium Rhodobacter sphaeroides. The disorder in LH1 appears to be dominated by the structural disorder, while that in LH2, by energetic one. These differences may be due to relatively bigger size of the LH1 complex and, consequently, with its enhanced structural flexibility.

Excitons in core LH1 antenna complexes of photosynthetic bacteria: Evidence for strong resonant coupling and off-diagonal disorder

The structure of the exciton manifold in the core bacteriochlorophyll antenna complexes from the purple photosynthetic bacterium Rhodobacter sphaeroides has been studied at 5 K using conventional and polarized fluorescence excitation spectroscopy supported with model simulations. Exciton bandwidths in the range of 2100–2200 cm −1, depending on sample integrity, have been determined in correlation with the largest mean coupling energy between the nearest-neighbor bacteriochlorophyll molecules close to 600 cm −1. These numbers are considerably bigger than those in any other native antenna complex known. Another noteworthy feature of these complexes is dominance of structural (off-diagonal) static disorder over the energetic (diagonal) disorder.

Bandwidth of excitons in LH2 bacterial antenna chromoproteins

The bandwidth of the exciton manifold in LH2 peripheral antenna complexes from the photosynthetic bacterium Rhodobacter sphaeroides has been determined using polarized fluorescence excitation spectroscopy and model simulations. It is shown that the fluorescence anisotropy spectra reveal a hidden structure of the exciton band that is correlated with its boundaries. The estimated exciton coupling energy and exciton bandwidth in the native complex is ∼360 and ∼1620 cm −1, respectively. The corresponding numbers in a mutant with the B800 bacteriochlorophyll molecules absent are somewhat larger (∼420 and ∼1890 cm −1), probably due to tightening of the protein structure.

Detection of zinc ions by differential circularly polarized fluorescence excitation.

A new chiroptical spectroscopic approach, differential circularly polarized fluorescence excitation (CPE), can be used to provide a selective method for detecting the presence of zinc ions. The approach utilizes the same instrumentation as fluorescence-detected circular dichroism and provides strong contrast in metal detection due to response to both chelation-enhanced fluorescence and circular dichroism upon metal ion binding. The observed contrast is therefore better than either of the parent spectroscopic detection methods. CPE also provides a strategy to reduce interference from background such as protein-based tryptophan fluorescence.

Excitation-and-collection geometry insensitive fluorescence imaging of tissue-simulating turbid media

We present an imaging technique for the correction of geometrical effects in fluorescence measurement of optically thick, turbid media such as human tissue. Specifically, we use the cross-polarization method to reject specular reflection and enhance the diffusive backscattering of polarized fluorescence excitation light from the turbid media. We correct the nonuniformity of the image field caused by the excitation-and-collection geometry of a fluorescence imaging system by normalizing the fluorescence image to the cross-polarized reflection image. The ratio image provides a map of relative fluorescence yield, defined as the ratio of emerging fluorescence power to incident excitation, over the surface of an imaged homogeneous turbid medium when fluorescence excitation-and-collection geometries vary in a wide range. We investigate the mechanism of ratio imaging by using Monte Carlo modeling. Our findings show that this technique could have a potential use in the detection of early cancer, which usually starts from a superficial layer of tissue, based on the contrast in the tissue fluorescence of an early lesion and of the surrounding normal tissue.

Evidence of pure Lb1 fluorescence from redshifted indole-polar solvent complexes in a supersonic jet

Two-photon polarized fluorescence excitation and vibronically resolved one-photon dispersed fluorescence spectra of the long-wavelength absorbing van der Waals complexes of indole with water, methanol, and formamide were examined for the purpose of firmly assigning the nature of the lowest singlet excited electronic state. The two-photon spectra for all of these complexes have circular/linear polarization ratios of absorptivity (Ω-values) that show that excitation is to the Lb1 state. Analysis of the Franck–Condon (FC) patterns of the dispersed fluorescence for these indole-polar solvent complexes show that emission is also from the Lb1 state in each case. In the emission spectra, the intensity ratios of the origin and ν26 lines are about 2:1, which is the value expected for Lb1 emission. The ν26 vibration is the most intense nonorigin vibronic line, as expected for Lb1 emission. Finally, there is little or no intensity from the ν8, ν9, and ν10 vibrations, which would be strongly active with emission from the La1 state. These results show unequivocally that for these indole-polar solvent complexes the La1 state does not shift in energy below the Lb1 state—contrary to the interpretation of several recent papers. Further support is given to the assignment of Lb1 emission by spectral simulations. The jet-cooled complexes do not exhibit excited state complex (exciplex) characteristics, wherein the solvent is much more strongly bound in the excited state than in the ground state.

Characterization of Salicylate-Alumina Surface Complexes by Polarized Fluorescence Spectroscopy

Speciation of salicylate anions (o-hydroxybenzoate) adsorbed on aqueous colloidal alumina (δ-Al2O3) was determined by polarized fluorescence excitation spectroscopy. Adsorption isotherms and pH and ionic strength edges suggest the existence of both inner-sphere and outer-sphere salicylate surface complexes. Spectroscopic characteristics of inner-sphere surface-salicylate complexes (one bidentate and two monodentate) were identified through comparison of suspension spectra with remarkably similar fluorescence and excitation spectra of solution phase Al-salicylate complexes. The large fluorescence Stokes gap of the aqueous salicylate anion is highly sensitive to complexation, resulting in spectral shifts characteristic of aluminum binding in the three inner-sphere salicylate complexes. These species appear to be present even at extremely low surface coverage, and the relative distributions are dependent on pH, ionic strength, and the relative concentrations of alumina and salicylate. The bidentate complex, however, is the predominant species at low surface coverages. Fluorescence anisotropy measurements, both steady-state and time-resolved, demonstrate that the bidentate and monodentate surface complexes do not undergo rotational reorientation on the time-scale of the fluorescence (τf = 4.0 ns), consistent with inner-sphere, polar covalent binding of these salicylate complexes to alumina surface sites. At high surface coverage, time-resolved anisotropy measurements suggest the existence of a surface salicylate species that is rotationally hindered (τr = 31 ps ) relative to free solution phase salicylate ions (τ r = 20 ps ). This behavior is consistent with an electrostatically bound outer-sphere complex suggested by the pH and ionic-strength sorption edges.

1La origin locations of methyl indoles in argon matrices

Polarized fluorescence excitation and dispersed fluorescence spectra of 3-methylindole (3MI), 5-methylindole (5MI), and 2,3-dimethylindole (2,3DMI) in solid Ar at 20 K under site selective conditions have unambigously revealed their 1La origin locations. For 3MI the 1La origin is 260 cm−1 above the 1Lb origin and for 5MI it is 1800 cm−1 above 1Lb. For 2,3DMI, the 1La origin lies 190 cm−1 below the 1Lb origin. The latter assignment is made from details of the dispersed fluorescence in comparison to the phosphorescence spectrum of indole in argon at 20 K.

The Discovery of a Novel R-phycoerythrin from an Antarctic Red Alga

A novel biliprotein, named R-phycoerythrin IV, has been discovered. It absorbs blue light better than any other known red algal biliprotein. The protein was found in Phyllophora antarctica, a benthic macroalga, which grows beneath the coastal waters of McMurdo Sound, Antarctica. Fluorescence emission and fluorescence excitation polarization spectroscopy demonstrated that R-phycoerythrin IV behaved as a typical R-phycoerythrin in the functioning of energy migration and has an emission maximum at 577 nm. The circular dichroism (CD) spectrum of the chromophores was compared with visible absorption spectrum, and both were deconvoluted. This process showed the energy states of various individual chromophores. The molecular weight of the protein suggested a alpha6beta6gamma polypeptide structure, and far UV CD studies revealed polypeptides with highly alpha-helical secondary structures. Dynamic light scattering indicated that the protein had a 5.54 nm radius, and its shape was nonspherical. R-phycoerythrin was also purified from a second benthic Antarctic red alga, Iridaea cordata. Its spectroscopic properties were similar to those of some R-phycoerythrins from nonpolar regions. The unique spectroscopic properties of R-phycocerythrin IV may help enable the alga to occupy its niche deeper in the water column than the red alga that has the typical R-phycoerythrin.

Aggregation of Chlorophyll a′ in Aqueous Methanol

ABSTRACTAn aggregate of chlorophyll a′ (Chi a′, C132‐epimer of ChI a) formed in a methanovwater (40160, vol/vol) mixed solvent was examined by visible absorption, circular dichro‐ism (CD), fluorescence and resonance Raman spectrosco‐pies in relation to its possible involvement in the core of photosystem I reaction center. The Chl a′ aggregate exhibited a sharp, double‐peaked absorption spectrum (690 and 715 nm) accompanied by an intense, conservative CD signal. The fluorescence excitation polarization spectrum showed that the doublet results from the exciton splitting in a single aggregate species. Time‐dependent changes in the spectroscopic properties clearly point to a simple transformation process from one molecular species to another, though a minor component appears to coexist. This conclusion was supported also by the principal multicom‐ponent spectral estimation analysis of the transients of absorption spectra. The species formed at the initial stage is most probably a T‐shaped dimer or oligomer, which is then gradually converted into the final major product, presumably a stacked dimer. In both of these states, the Chl molecules are linked together via direct coordination of the C13’keto carbonyl oxygen onto the Mg atom of neighboring molecules, as suggested by almost identical resonance Raman spectra in ordinary and deuterated methanovwater mixed solvents. The stacked dimers probably further associate to form a colloidal state in this solvent system. Based on these results, a model for the Chl a′ aggregation is proposed.

Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin.

Distance relationships between phalloidin binding sites on F-actin have been investigated using fluorescence resonance energy transfer (FRET) techniques in solution and on single F-actin filaments bound to heavy meromyosin (HMM). Filaments saturated with an equimolar concentration of fluoresceinisothiocyanatophalloidin (FITC-ph) as the donor and tetramethylrhodamineisothiocyanatophalloidin (TRITC-ph) as the acceptor and control filaments saturated with either FITC-ph or TRITC-ph were characterized by absorption and fluorescence spectroscopy and the in vitro motility assay. Fluorescence excitation polarization spectroscopy showed hetero-FRET occurred within colabeled filaments, whereas homo-FRET was observed in control filaments. The distance measured between adjacent phalloidin binding sites using randomly labeled FITC-ph and TRITC-ph was 37.2 A using steady-state spectral analysis and 36.9 A using time-resolved spectroscopy with a radial coordinate of 14.5 A. Measurements of the distance between fluorescent phalloidin groups using the atomic model of F-actin [Lorenz, M., Popp, D., & Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836] suggest transfer occurs between adjacent phalloidin molecules on opposite actin helices (39 A), rather than between adjacent phalloidins along the same actin helix (55.4 A). A quantitative fluorescence microscope technique was described that measures the proximity of adjacent FITC-ph and TRITC-ph on single filaments immobilized on HMM. Here a distance of 36.2 A was calculated which was unchanged during ATP-dependent sliding of F-actin on HMM. Spatially resolved FRET measurements are being used to observe the effects of actin binding proteins on the structural properties along the length of single actin filaments.

Site selective photoselection study of indole in argon matrix: location of the 1L a origin

Polarized fluorescence excitation and dispersed fluorescence spectra of indole in solid Ar at 20 K under site selective conditions have revealed that ν 28 is Herzberg-Teller (HT) active, both in the excitation and the fluorescence spectrum. The two lines 455 and 480 cm −1 above the 1L b origin are a Fermi doublet involving 28 0 1 and the out-of-plane combination 39 0 141 0 1 and derive 1L a optical character via the HT coupling by ν 28. The true 1L a origin appears to be about 1400 cm −1 above the 1L b origin in vapor, as discerned by the differential red shift in the Ar matrix.

Incorporation of stilbazolium merocyanines into resting and stimulated mononuclear leukocytes.

Stimulated and resting mononuclear leukocytes were incubated with a stilbazolium merocyanine dye 1-(6'-hydroxyhexyl)-4-[(4-oxocyclohexa-2,5- dienylidene)ethylidene]-1,4-dihydropyridine and immobilized in isotropic and stretched polyvinyl alcohol film. Polarized absorption, fluorescence and fluorescence excitation spectra were collected and the anisotropy of absorption and emission were calculated. Analysis of the spectra pointed to: i. the occurrence of perturbation of the membrane structure by incubation with the dye, and ii. influence of the blood serum addition, during the process of incubation with the dye, on the efficiency of incorporation of merocyanine into the cells and the degree of the dye orientation in the membrane. A small fraction of the dye molecules introduced into resting cells was found oriented to a higher degree than a large fraction incorporated into stimulated cells. The incubation time longer than 15 min caused strong changes in the membrane structure both of the resting and stimulated cells.