Orientation Of Transition Dipoles Research Articles

Mapping the orientation of exciton transition dipoles along individual nanostructures of molecular J-aggregates

Modulation of the polarization state of incident light in reflection microspectroscopy is used to study locally the orientations of exciton transition dipole moments of fiberlike nanostructures of J-aggregates of pseudoisocyanine dyes. The lowest exciton state assumes a wide range of directions with respect to the long axis of the fibers, from parallel to perpendicular. This behavior is correlated with the onset of polariton-like character of the local reflectance spectra. The results imply that a new structural model has to be conceived to explain the observed phenomena.

Determination of Molecular Orientation in Self-Assembled Monolayers Using IR Absorption Intensities: The Importance of Grinding Effects

The orientation of organic chain molecules grafted to a metal surface is commonly determined using infrared reflection absorption spectroscopy (IRRAS). Whereas the acquisition of the IR spectrum itself is rather straightforward, the determination of orientational parameters (tilt angle, twist angle) is complicated by the requirement to use the bulk reference spectrum of the substance taken to form the self-assembled monolayer (SAM). In the past, mainly two methods have been used to extract orientational information from IR data. The application of the absolute method requires the precise knowledge of the complex refractive index, but the intensities of the IR bands are influenced by Mie scattering of light in the pellet which depends on the particle size and shape and therefore on the grinding time and conditions. On the other hand, the application of the method using relative intensities requires a second IR-active vibration with a differently oriented transition dipole moment (TDM) to eliminate the rela...

Z-polarized confocal microscopy.

In light microscopy the transverse nature of the electromagnetic field precludes a strongly focused longitudinal field component, thus confining polarization spectroscopy and imaging to two dimensions (x,y). Here we describe a simple confocal microscopy arrangement that optimizes for signal from molecules with transition dipoles oriented parallel to the optic axis. In the proposed arrangement, we not only generate a predominant longitudinally (z) polarized focal field, but also engineer the detection scheme in such a way that in a bulk of randomly oriented molecules, the microscope's effective point-spread function is dominated by the contribution of those molecules that are oriented along the optic axis. Our arrangement not only implicitly allows for the determination of the orientation of transition dipoles of single molecules in three dimensions, but also highlights the contribution of z-oriented molecules in three-dimensional imaging.

Z-polarized confocal microscopy

In light microscopy the transverse nature of the electromagnetic field precludes a strongly focused longitudinal field component, thus confining polarization spectroscopy and imaging to two dimensions (x,y). Here we describe a simple confocal microscopy arrangement that optimizes for signal from molecules with transition dipoles oriented parallel to the optic axis. In the proposed arrangement, we not only generate a predominant longitudinally (z) polarized focal field, but also engineer the detection scheme in such a way that in a bulk of randomly oriented molecules, the microscope's effective point-spread function is dominated by the contribution of those molecules that are oriented along the optic axis. Our arrangement not only implicitly allows for the determination of the orientation of transition dipoles of single molecules in three dimensions, but also highlights the contribution of z-oriented molecules in three-dimensional imaging.

The Effect of Correlation of Inhomogeneous Environmental Shifts on Raman Depolarization Ratio Dispersion

The magnitude and sense of correlation between the inhomogeneous broadening distributions of two neighboring electronic states is shown to have a significant effect on the dispersion of the depolarization ratio, ρ, for Raman scattered radiation. To examine this effect quantitatively we treat numerically a model in which two electronic excitations with mutually perpendicular transition dipole orientations are represented by shifted one-dimensional harmonic potentials. The inhomogeneous broadening is treated in the static limit as a Gaussian fluctuation in each of the two electronic origins. The correlation of these shifts is treated as a variable with the fully correlated and fully anti-correlated limits spanning the uncorrelated case. It is shown that the degree of correlation (and its sign) strongly influences the value of the depolarization ratio in some cases which should be amenable to experimental study. Three cases are treated which differ in the separation of the electronic origins of the two state...

Self-waveguided blue light emission in p-sexiphenyl crystals epitaxially grown by mask-shadowing vapor deposition

Highly polarized and self-waveguided blue light emission was observed in oriented organic crystals of p-sexiphenyl (6P). The 6P molecules were epitaxially grown in needlelike crystals with a submillimeter length by mask-shadowing vapor deposition on the KCl (001) surface. In the crystals, the molecular axes are oriented parallel to the [110] direction of the KCl substrate and perpendicular to the needle axis of the crystal. Under ultraviolet excitation, the blue light emission was confined within the crystal and self-waveguided along the needle axis with the transverse-electric mode, then radiated from the tips of the needles. This self-waveguided emission in the crystal was based on the uniaxially oriented transition dipoles which lay on the KCl surface and were aligned perpendicular to the needle axis in the crystal.

Protein S Alters the Active Site Location of Activated Protein C above the Membrane Surface

The location of the active site of membrane-bound activated protein C (APC) relative to the phospholipid surface was determined both in the presence and absence of its cofactor, protein S, using fluorescence resonance energy transfer (FRET). APC was chemically modified to create the FRET donor species, Fl-FPR-APC, with a fluorescein dye (Fl) covalently attached to the active site via a D-Phe-Pro-Arg (FPR) tether and located in the active site near S4. FRET was observed when Fl-FPR-APC was titrated in the presence of Ca2+ ions with phosphatidylcholine/phosphatidylserine (4:1) vesicles containing the FRET acceptor, octadecylrhodamine (OR). Assuming a random orientation of transition dipoles (kappa2 = 2/3), the average distance of closest approach between the fluorescein in the active site of the membrane-bound APC and the OR at the membrane surface is 94 A. The same calcium-dependent distance was obtained for both small and large unilamellar vesicles and for vesicles that contained phosphatidylethanolamine. The active site of membrane-bound APC is therefore located far above the phospholipid surface. Upon addition of protein S, the efficiency of Fl-FPR-APC to OR energy transfer increased due to a protein S-dependent rotational and/or translational movement of the APC protease domain relative to the surface. If this movement were solely translational, then the average height of the fluorescein in the membrane-bound APC.protein S complex would be 84 A above the surface. The extent of Fl-FPR-APC to OR energy transfer was unaltered by the addition of thrombin-inactivated protein S. The protein S effect was also specific for APC, since the addition of protein S to similarly-labeled derivatives of factor Xa, factor IXa, or factor VIIa did not alter the locations of their active sites. This direct measurement demonstrates that the binding of the protein S cofactor to its cognate enzyme elicits a relocation of the active site of APC relative to the membrane surface and thereby provides a structural explanation for the recently observed protein S-dependent change in the site of factor Va cleavage by APC.

Femtosecond Four-Wave Mixing Spectroscopy of Molecular Aggregates

Two-photon resonance-enhanced four-wave mixing processes taking place in circular aggregates are considered theoretically with respect to their influences on pump−probe difference absorption measurements. Remarkable changes of the signal spectra around the zero time delay arising from the two-photon resonance transitions to the two-exciton states were discovered. Time- and frequency-resolved pump−probe measurements in the vicinity of zero delay times were shown to be sensitive to the manifestation of two-exciton states and, consequently, may be used for the analysis of the transition dipole orientation of the individual molecules as well as the extent of exciton localization in the circular aggregates.

Supramolecular Assemblies of Chromophores in LB Films and Related Media

Formation of aggregates has been observed as a general phenomenon for a wide variety of organic molecules, especially aromatic compounds and dyes. Aggregation is most commonly encountered in crystals or in thin films. However, it has been increasingly observed in microheterogenous media or in other situations where high local concentrations occur or where specific orientation is favored. Two limiting types of aggregation have been defined based on the orientation of transition dipoles and their absorption spectral characteristics. These are the “J” aggregate, in which head-to-tail arrangements of transitiondipole moments are characterized by a sharp, intense, red-shifted transition compared to the isolated (solvated) monomer, and the “H” aggregate, where head-to-head (card-pack) orientations are characterized by a blue shift of the prominent transition compared with the monomer. Several treatments have been proposed to correlate the observed spectral shifts with the aggregate structure. For a number of compounds, the association of known x-ray-determined structures with spectral features has supported the theoretical predictions developed by Kasha and Hochstrasser or by Czikkely, Försterling, and Kuhn. The focus of the studies described here has been on aggregation occurring in Langmuir-Blodgett (LB) films and related media, such as bilayer vesicles, which are characterized by an assembly of molecules in an interfacial situation where a polar-nonpolar or water-hydrocarbon boundary should provide a strong organizing influence. In early cases where aggregates were encountered as prominent components of mixed LB films (even when relatively dilute mixtures were used), the phenomenon was usually dismissed as “microcrystallization,” which was considered an unavoidable nuisance and not really due to fundamental intermolecular interactions. More recent studies have shown that aggregation in these media is really a significant molecular phenomenon that shows dependence both on the specific molecules and the topology of the film-forming surfactant. Although some previous investigations have been carried out with different results for various substrates, we have embarked on a study to correlate aggregation behavior for a number of different chromophores incorporated into amphiphilic structures to obtain a general picture of the relative importance of different factors that can control aggregation phenomena.

Femtosecond spectral and anisotropy study of excitation energy transfer between neighbouring α-80 and β-81 chromophores of allophycocyanin trimers

Polarization pump-probe femtosecond spectroscopy was used to investigate photoinduced optical density changes in allophycocyanin (APC) trimers at 635–690 nm after excitation with 230-fs pulses at 618 nm. The initial bleaching observed at λ < 645 nm is followed by subpicosecond absorption recovery corresponding to 430 ± 40 fs recovery kinetics measured at 615 nm with 70-fs pulses. Only the red part of the APC absorption band remains strongly bleached at 3 ps after excitation. The spectral and kinetic results can be described in terms of two different models of interaction between neighbouring α-80 and β-81 chromophores of APC trimers. According to the first one, the observed subpicosecond kinetics corresponds to relaxation between the levels of excitonically coupled, spectrally identical α-80 and β-81 chromophores. Excited state absorption to doubly excited excitonic state should in this case contribute to the measured difference spectra. According to the second one, the femtosecond excitation energy transfer in APC trimers takes place between a donor chromophore absorbing predominantly at 620 nm and an acceptor chromophore absorbing at 650 nm. The high anisotropy value observed at 615 nm during the first 1.2 ps is in good agreement with the donor-acceptor model. Anisotropy values calculated in the 635–675 nm spectral region at 3 ps after excitation are in the 0.1–0.25 range corresponding to an angle of 30°–45° between donor and acceptor transition dipole orientations. The high anisotropy obtained at 658 nm during the excitation is probably due to stimulated emission of the donor chromophore.

Orientation of actin filaments during motion in in vitro motility assay

Rhodamine-phalloidin was added to F-actin, and the orientation of transition dipoles of the dye was measured in single actin filaments by polarization of fluorescence. Rhodamine-phalloidin was well immobilized on the surface of actin, indicating that changes in orientation of the dye reported changes in orientation of actin monomers. In stationary filaments the dipoles were inclined at 49.3 degrees with respect to the filament axis. The disorganization of dipoles in stationary filaments was insignificant. When the filaments were made to translate, the average orientation of the dye did not change, but disorganization slightly increased. Disorganization increased significantly when filaments were free in solution. We concluded that, within the accuracy of our measurements (approximately 18%), actin monomers did not undergo major reorientations during motion, but that binding of myosin heads deformed the structure of filaments.

Protein side chain rotational isomerization: A minimum perturbation mapping study

A theory of the rotational isomerization of the indole side chain of tryptophan-47 of variant-3 scorpion neurotoxin is presented. The isomerization potential energy, entropic part of the isomerization free energy, isomer probabilities, transition state theory reaction rates, and indole order parameters are calculated from a minimum perturbation mapping over tryptophan-47 χ1×χ2 torsion space. A new method for calculating the fluorescence anisotropy from molecular dynamics simulations is proposed. The method is based on an expansion that separates transition dipole orientation from chromophore dynamics. The minimum perturbation potential energy map is inverted and applied as a bias potential for a 100 ns umbrella sampling simulation. The entropic part of the isomerization free energy as calculated by minimum perturbation mapping and umbrella sampling are in fairly close agreement. Throughout, the approximation is made that two glutamine and three tyrosine side chains neighboring tryptophan-47 are truncated at the Cβ atom. Comparison with the previous combination thermodynamic perturbation and umbrella sampling study suggests that this truncated neighbor side chain approximation leads to at least a qualitatively correct theory of tryptophan-47 rotational isomerization in the wild type variant-3 scorpion neurotoxin. Analysis of van der Waals interactions in a transition state region indicates that for the simulation of barrier crossing trajectories a linear combination of three specially defined dihedral angles will be superior to a simple side chain dihedral reaction coordinate.

Electronic energy transfer in anisotropic systems. III. Monte Carlo simulations of energy migration in membranes

Electronic energy migration in membrane systems has been studied by means of Monte Carlo (MC) simulations according to an algorithm described previously [S. Engström, M. Lindberg, and L. B.-Å. Johansson, Chem. Phys. 89, 204 (1988)]. In the systems investigated, the interacting donor molecules are randomly localized in mono-, bi-, and multilayers and are either oriented with their transition dipoles isotropically or parallel to the layers. The mean-square displacement [〈R(t)2〉] of the excitation and experimental observables in terms of different fluorescence depolarizations were determined. All results are relevant for the ‘‘slow case,’’ which means that translational and rotational motions of the donors are much slower as compared to the rate of fluorescence. A two-particle approximation for calculating the excitation probability of the initially excited molecule [denoted by Gs(t) ] and the fluorescence depolarizations in two-dimensional systems has been published previously [J. Baumann and M. D. Fayer, J. Chem. Phys. 85, 4087 (1986)]. By using the MC algorithm, we have in this work tested this model extensively. The excitation probability Gs(t) is found to be in excellent agreement with the MC simulations for all of the systems studied. For isotropically oriented donor molecules in multilamellar systems, the simulations show that Gs(t) is very well approximated by that of a monolayer at distances of d≥3 R0 between the layers. At distances of d≤0.5 R0, the function Gs(t) is equal to that of a three-dimensional solution. For the in-plane oriented dipoles in a multilayer system, Gs(t) is very well approximated by that for a single bilayer at d&amp;gt;2 R0. In general, the depolarizations obtained by the two-particle model and MC simulations differ depending on the particular orientational distribution and the experimental geometry. To obtain a physically correct behavior of the fluorescence anisotropy at long times (i.e., the limiting anisotropy) is not possible within a two-particle approach. We instead propose a phenomenological model for calculating the fluorescence anisotropy which is a function of Gs(t) obtained within the two-particle approximation. This model gives a remarkably good agreement with the MC simulations and has the correct limiting anisotropy. The MC simulations of energy migration show that 〈R(t)2〉 is about twice as large for in-plane oriented donors in mono- and bilayers, as compared to the case of isotropically oriented transition dipoles. For multilayer systems consisting of layers separated by d=R0, we find that 〈R(t)2〉 is only slightly larger for the in-plane oriented transition dipoles as compared to the isotropic orientational distribution.

Orientation of xanthophylls in phosphatidylcholine multibilayers

Oriented multibilayers of dimyristoyl phosphatidylcholine (DMPC) modified with violaxanthin or zeaxanthin were examined by X-ray diffractometry and linear dichroism. It appears that pigment molecules and the normal to the bilayer plane form an angle of 24–25°. It was also observed that rather small concentrations of added xanthophylls (molar fraction up to 3%) increase the pigmented bilayer thickness by a value of about 2 Å as compared with that of the pure DMPC bilayer. The observed nonzero linear dichroism at normal incidence of light suggests the possibility of nonhomogeneous orientation of transition dipoles in the plane of the bilayer.

Transition dipole orientations in the early photolysis intermediates of rhodopsin

The linear dichroism spectrum of rhodopsin in sonicated bovine disk membranes was measured 30, 60, 170, and 600 ns after room temperature photolysis with a linearly polarized, 7-ns laser pulse (lambda = 355 or 477 nm). A global exponential fitting procedure based on singular value decomposition was used to fit the linear dichroism data to two exponential processes which differed spectrally from one another and whose lifetimes were 42 +/- 7 ns and 225 +/- 40 ns. These results are interpreted in terms of a sequential model where bathorhodopsin (BATHO, lambda max = 543 nm) decays toward equilibrium with a blue shifted intermediate (BSI, lambda max = 478 nm). BSI then decays to lumirhodopsin (LUMI, lambda max = 492 nm). It has been suggested that two bathorhodopsins decay in parallel to their products. However, a Monte Carlo simulation of partial photolysis of solid-state visual pigment samples shows that one mechanism which creates populations of BATHO having different photolysis rates at 77 K may not be responsible for the two decay rates reported here at room temperature. The angle between the cis band and 498-nm band transition dipoles of rhodopsin is determined to be 38 degrees. The angles between both these transition dipoles and those of the long-wave-length bands of BATHO, BSI, and LUMI are also determined. It is shown that when BATHO is formed its transition dipole moves away from the original cis band transition dipole direction. The transition dipole then moves roughly twice as much towards the original cis band direction when BSI appears. Production of LUMI is associated with return of the transition dipole almost to the original orientation relative to the cis band, but with some displacement normal to the plane which contains the previous motions. The correlation between the lambda max of an intermediate and its transition dipole direction is discussed.

Quantitative method for studying orientation of transition dipoles in membrane vesicles of spherical symmetry

Pigmented vesicular membranes embedded in polyacrylamide gel exhibit linear dichroism when the gel sample is squeezed [Abdourakhmanov, I.A., Ganago, A.O., Erokhin, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979)_Biochim. Biophys. Acta 546, 183–186]. The orientation technique of gel-squeezing was modified to enhance polarization effects in membrane vesicles of spherical symmetry. Model calculations were carried out to provide a tool for the quantitative evaluation of the dichroism of squeezed gel samples. The orientation angles of the dipoles can be calculated with reasonable precision by measuring two quantities: (i) the macroscopic deformation parameter of the gel sample, and (ii) a parameter (e.g. the polarization ratio of the fluorescence emission) characterizing the orientation of the transition dipoles in the membranes embedded in the squeezed gel. The validity of the model was confirmed through a series of polarization measurements relating to the fluorescence of chlorophyll a in membranes of osmotically shocked chloroplasts, ‘blebs’.

Picosecond study of energy-transfer kinetics in phycobilisomes of Synechococcus 6301 and the mutant AN 112

Excitation-energy-transfer kinetics in isolated phycobilisomes from the cyanobacterium Synechococcus 6301 ( Anacystis nidulans) and the mutant AN 112 (rods containing one hexameric C-phycocyanin unit only) was investigated by picosecond absorption and fluorescence techniques. The different chromophores in the phycobilisomes were selectively excited. A lifetime component of about 10 ps was found for both C-phycocyanin and allophycocyanin in both types of phycobilisomes. We assign these signals to a transfer of excitation energy from sensitizing (‘s’) to fluorescing (‘f’) chromophores within C-phycocyanin and allophycocyanin units. A 10 ps component was also observed in the anisotropy relaxation measurements. The anisotropy decay is attributed mainly to differently oriented transition dipole moments of ‘s’- and ‘f’-chromophores and partially to ‘f’ → ‘f’ transfer. An absorption recovery signal of τ ≈ 90 ps at λ ≤ 630 nm in phycobilisomes of Synechococcus 6301 is reduced to 40–50 ps in AN 112 phycobilisomes. This is rationalized in terms of a decreased rod → core transfer time in the shorter rods of AN 112. The 40–50 ps lifetime of fluorescence and absorption recovery in AN 112 phycobilisomes is assigned mainly to a rate-limiting transfer step between C-phycocyanin and the allophycocyanin core. A decay component of allophycocyanin τ ≈ 50 ps was observed both in absorption recovery measurements and in fluorescence decay. It is assigned to energy transfer to the terminal chromophores. The final emitter(s) of the phycobilisomes from AN 112 have fluorescence lifetimes of 1.9 and 1.3 ns. We find a good correlation in the fluorescence kinetics between the decay times of phycocyanin and allophycocyanin and the fluorescence risetimes of the terminal emitters.

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.

Antenna chlorophyll a and P700. Exciton transitions in chlorophyll a arrays

Exciton calculations have been carried out for 16 distinct cases of randomly generated chlorophyll a arrays to assist in the assessment of various possibilities for the chlorophyll distribution in the thylakoid membrane of green plants. Each of the cases is distinguished by its packing density and by the constraints applied to the orientation of transition dipoles. Each array consists of 40 antenna chlorophyll a molecules with an absorption maximum at 670 nm before transition density coupling and one P700 chromophore with an absorption maximum at 700 nm. Transition density coupling always broadens the absorption bands and sometimes shifts the position of the absorption maxima. Transition density coupling between the P700 chromophore and the antenna chlorophyll molecules shifts the P700 absorption band to lower energies and, for some cases, increases or decreases the dipole strength of the P700 absorption band by as much as 50 percent. In general, for comparable packing densities, the effects of transition density coupling are more pronounced for two-dimensional arrays than for three-dimensional arrays. The present exciton calculations show that if the various in vivo ''forms'' of chlorphyll a correspond to exciton transitions in chlorophyll a arrays, then the local chlorophyll concentration in the arrays must bemore » much greater than the average chlorophyll concentration of approximately 0.1 M in the thylakoid membrane; perhaps the local chlorophyll concentration could be as high as 1 M.« less

Interpretation of energy-transfer experiments by theoretical studies of model compounds using semiempirical potential functions. I. Three-linked aromatic pepide units.

AbstractIt is shown that theoretical conformational analysis, based on the evaluation of semiempirical potential functions, can be used to compute the quantities relevant to the interpretation of energy‐transfer experiments. The relevant properties are computed for a segment of a polypeptide chain with the sequence Tyr‐Tyr. In particular, the average value of the orientation factor 〈κ2〉 and its distribution ƒ(κ2) are examined. It appears that the degrees of freedom for rotation of the side chains are not sufficient to randomize completely the orientation factor of the transition dipoles. Two additional degrees of freedom, namely the torsion angles around the valence bonds of the backbone, ψ1 and ϕ2, bring 〈κ2〉 close to the value that corresponds to randomly oriented transition dipoles.