Time-resolved Fluorescence Depolarization Technique Research Articles

Rotational dynamics study of structurally similar (medium sized) coumarin dyes in aqueous mixtures of DMSO and DMA

The rotational diffusion dynamics of four medium sized, structurally similar, polar Coumarin dyes namely, Coumarin-478 (C478), Coumarin-480 (C480), Coumarin-519 (C519), and Coumarin-523 (C523) has been studied at room temperature in aqueous DMSO and aqueous DMA by using steady state and time resolved fluorescence depolarization techniques. All the probes exhibited faster rotation in organic solvent rich zone than in water rich zone that resulted in a downward bent “hook profile” of rotational reorientation time as a function of viscosity. A faster rotational diffusion is observed for C523 in contrast to the other three solutes in both the aqueous solvent mixtures. The difference between τr values at iso-viscous points is smaller in case of aqueous DMA than in aqueous DMSO which reveals the importance of dielectric friction. Since both solutes and solvents are polar in nature, the Nee-Zwanzig (NZ) and van der Zwan and Hynes (ZH) dielectric friction theories have been used along with Stokes-Einstein-Debye (SED) hydrodynamic theory to assess the experimental results and understand the microfriction experienced by the solute. An important aspect of this work is to establish a relation between viscosity and hydrogen bond strengths at varying compositions of solvent mixtures as well as hydrogen bond strength between solute and solvents with the help of Gaussian 09 software. The H-bond strength thus calculated is found to be higher at a composition where the viscosity is the maximum. It provides an additional support to the experimental results.

Solvation, rotational dynamics, photophysical properties study of aromatic asymmetric di-ketones: An experimental and theoretical approach

In this article we have investigated the solvation, rotational dynamics and photo physics of diketone derivatives DK-2 and DK-3 in alcohols and alkanes at room temperature through both a time-resolved fluorescence depolarization technique and steady state. Viscosity of dissolvable varies linearly with respect to rotational times of the molecule. Outcomes are investigated by utilizing the theory of hydrodynamic (SED) Stokes- Einstein-Debye to evaluate the friction with stick and slip limiting conditions. In addition, using the solvatochromic shift and quantum chemical technique aromatic di-ketone derivatives, 1-(4′-dodecyl-[1,1′-biphenyl]-4-yl)-3-(4-ethoxyphenyl) propane-1,3-dione (DK-2) and 1-(4′-dodecyl-[1,1′-biphenyl]-4-yl)-3-(4-(nonyloxy)phenyl)propane-1,3-dione (DK-3) estimated properties of fluorescence. The excitation and emission spectra of DK-2 and DK-3 have been observed in several solvents. The dipole moments calculated using DFT theory and solvatochromic correlations. Estimated (μg) value by DFT theory is relatively greater than the solvatochromic shift techniques. The dipole moments of excited state (μe) are found to be more prominent than dipole moment of ground states (μg) which approves π → π* transition. The variance of dipole moments (Δμ) was determined from microscopic solvent polarity parameter (ETN) and solvatochromic shift technique and outcomes are compared.

Rotational Diffusion of a New Large Non Polar Dye Molecule in Alkanes.

Rotational reorientation times of a newly synthesized 2,5-bis(phenylethynyl)1,4-bis(dodecyloxy) benzene (DDPE) are experimentally determined in series of n-alkanes by employing steady state and time resolved fluorescence depolarization technique with a view to understand rotational dynamics of large non-polar solute molecule in non-polar solvents and few general solvents of different sizes and varying viscosity. It is observed that rotational reorientation times vary linearly as function of viscosity. The hydrodynamic stick condition describes the experimental results at low viscosities while the results tend to deviate significantly from it at higher viscosities. This is attributed to the possibility of long chains in solvents hosting a variety of chain defects (end-gauche, double-gauche, all-trans, kink, etc.) thereby reducing the effective length of the molecule, leading to a slightly reduced friction. The experimental results are compared with the predictions of Stokes-Einstein-Debye (SED) hydrodynamic theory as well as the quasi-hydrodynamic theories of Gierer-Wirtz (GW) and Dote-Kivelson-Shwartz (DKS). The predictions from these theories underestimate τr in the solvents employed in the study.

Influence of temperature on rotational diffusion of dipolar laser dyes in glycerol

Temperature dependent rotational diffusion of three medium sized dipolar laser dyes viz., Fluorescein 27 (F27), Fluorescein Na (FNa) and Sulforhodamine B (SRB) has been studied in glycerol using both steady-state and time-resolved fluorescence depolarization techniques. The rotational reorientation times of these probes are observed to vary linearly as a function of viscosity over the range of temperature studied. The results have been discussed and analyzed in the light of Stokes–Einstein–Debye (SED) hydrodynamic theory. The reorientation times of the probes follow a trend that is well within hydrodynamic slip and stick limits. Finally, the applicability of quasihydrodynamic models of Geirer–Wirtz (GW) and Dote–Kivelson–Schwartz (DKS) is discussed with a view to understand the nature of solute–solvent interactions.

Translational and rotational motions of proteins in a protein crowded environment

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer–crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.

Solvent Dependence of the Hydrodynamical Volume of Dendrimers with a Rubicene Core

Four generations of a dendrimer with a fluorescent core consisting of a rubicene moiety are synthesized. The biexponential nature of fluorescence decay in toluene indicates the presence of two emitting conformations. Molecular modeling suggests that a conformation where the dendrons interact with the core is not improbable. The relative weight of the two decay times indicates that the contribution of that conformation in toluene increases as the dendrimer generation increases. In acetone and acetonitrile however the fluorescence decay is, except for the fourth generation, monoexponential. The hydrodynamical volume of the dendrimer is determined in toluene, a good solvent, acetone, a medium quality solvent, and acetonitrile, a poor solvent, with the time-resolved fluorescence depolarization technique. No change of the hydrodynamical volume is found in toluene in a temperature range between 20 and 94 °C. This suggests that the dendrimers of all the generations are fully expanded in this solvent. In acetonitrile however the hydrodynamical volume of the dendrimers is substantially smaller than in toluene and acetone. This effect is most clear for the fourth-generation dendrimer. For this compound the hydrodynamical volume is only a few percentages larger than the excluded volume.

Fluidity of glycerol skeletal region in phospholipid bilayers: a time-resolved fluorescence depolarization study.

The fluidity of glycerol skeletal region in phospholipid bilayer was investigated by the time-resolved fluorescence depolarization technique with L-alpha-dihexadecanoyl-sn-glycero-3-phospho-[N-(4-nitrobenzo-2-oxa-1,3- diazole)]ethanolamine (NBD-PE) as a fluorescent probe. In this probe, the fluorescent moiety, 4-nitrobenz-2-oxa-1,3-diazole (NBD), is attached to a nitrogen atom at the polar head group of phosphatidylethanolamine molecule. When this probe is embedded in a lipid bilayer, the NBD moiety locates near the glycerol skeletal region. The time courses of fluorescence anisotropy of NBD-PE in dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers were analyzed using a wobbling-in-cone model, in which the molecular motion is characterized by a half cone angle (theta c) and a wobbling diffusion rate (Dw). Values of Dw of NBD moiety in phospholipid bilayers were found to be on the order of 10(7) s-1 at the physiological temperatures, which is almost the same value as that of the hydrocarbon chain in lipid bilayers. This fact indicates that the fluidity in the glycerol skeletal region is similar to that in the hydrocarbon layer.

Multichromophoric cyclodextrins. 1. Synthesis of O-naphthoyl-.beta.-cyclodextrins and investigation of excimer formation and energy hopping

The synthesis and photophysical properties of /3-cyclodextrins bearing seven 2-naphthoyloxy chromophores in specific positions, either on the primary face or the secondary face, or 14 2-naphthoyloxy chromophores, seven on each face, are reported. These multichromophoric cyclodextrins are good models for the study of excitation energy migration among chromophores in well-defined positions. The investigation was performed in dichloromethane and in a mixture of ethanol and methanol that can form a glass at low temperature. The absorption spectra show that the interactions between chromophores in the ground state are weak, whereas the fluorescence spectra reveal the existence of excimers at room temperature but not at low temperature in a rigid glass. Further evidence of excimer formation is provided by the fluorescence decays. Since excimers act as energy traps, the energy hopping process was studied in a rigid glass at low temperature by steady-state and time-resolved fluorescence depolarization techniques. The steady-state anisotropy is found to be one seventh of the theoretical limiting anisotropy 0.4, which means that excitation energy hops between chromophores with essentially randomly oriented transition moments at a rate much higher than the chromophore intrinsic decay rate. Energy hopping is indeed very fast as shown by the fluorescence anisotropy decay which is at least as fast as the apparatus time resolution (a few tens of picoseconds).

Rotational resolution of methyl-group substitution and anisotropic rotation of flavins as revealed by picosecond-resolved fluorescence depolarization

The rotational correlation times of the natural flavin compounds riboflavin and FMN and the flavin models lumiflavin, tetra-O-acetyl-riboflavin and their N (3)-methylated derivatives, dissolved in water, were determined with the time-resolved fluorescence depolarization technique. Owing to the time resolution the effect of methyl group substitution could be determined as a few picoseconds longer correlation time, in agreement with the slightly larger molecular volume. Anisotropic rotation of lumiflavin was demonstrated by observing the change in correlation time when excitation was in the second excited singlet state of the flavin. The angle between the emission transition moment and the long axis of the flavin ring system is approximately 22°.

Membrane viscosity of lymphocytes and influence of phytohemagglutinin

The membrane viscosity of peripheral blood lymphocytes (PBLs) of equine, bovine and canine was measured by the use of time-resolved fluorescence depolarization technique with 1, 6-diphenyl-1,3,5-hexatriene (DPH). The viscosity values were 0.55, 0.59 and 0.50 poise for equine, bovine and canine PBLs, respectively. These values were compared with steady-state anisotropies and order parameters measured from electron spin resonance (ESR) of 5-doxyl stearic acid. Both values were increased with increase of viscosity. The fluid property of the membranes stimulated with phytohemagglutinin-P (PHA) was measured with steady-state fluorescence anisotropy and ESR. Little change of membrane fluidity was recognized with both methods during the stimulation with PHA. It appears that PHA activation process for these lymphocytes does not included large increase of the membrane fluidity which significantly accelerate the diffusion velocity of receptors in the plasma membrane.

A Comparison of the Molecular Dynamics of Fluorescent Probes in Curved Lipid Vesicles and Planar Multibilayers

Abstract The dynamic behaviour of the probe molecules DPH and TMA-DPH embedded in small unilamellar vesicles and planar multibilayers of POPC has been studied by time-resolved fluorescence depolarization techniques. The molecular dynamics of the probe molecules was analysed in terms of the rotational diffusion model. It is found that analysis of the time-dependent fluorescence anisotropy from the vesicle system yields two distinct, though statistically equivalent solutions. On the other hand the measurements on planar multibilayers can be interpreted unequivocally. It is shown that the order parameters of the probe molecules are higher in the multibilayers than in the vesicles. A reconstruction of the orientational distribution function reveals that the TMA-DPH molecules have a more pronounced tendency to lie with their long axes parallel to the bilayer surface in the curved vesicles than in the planar multibilayers. An intriguing finding is that the reorientational motion of the probes is considerably sl...

Time-resolved fluorescence depolarization techniques in model membrane systems. Effect of sterols and unsaturations.

Application of time-resolved fluorescence depolarization techniques to the study of biological membrane structure and dynamics requires selective signal monitoring of specifically located intrinsic or extrinsic fluorophores. In such systems, at least to date, the fluorescence characteristics of the naturally occurring chromophores (e.g., aromatic amino acid residues, enzymatic reactional cofactors, polyenic fatty acids and sterols, chlorophyll, and retinal) are of limited use because of low and/or reabsorbed emitted intensities, low signal-to-noise ratio (e.g., light scattering), and so on.KeywordsAcyl ChainFluorescence AnisotropyFatty Acyl ChainFluorescence DepolarizationParinaric AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.