Fluorescence Depolarization Experiments Research Articles

Probing the Local Nanoscale Heating Mechanism of a Magnetic Core in Mesoporous Silica Drug-Delivery Nanoparticles Using Fluorescence Depolarization.

In the presence of an alternating magnetic field (AMF), a superparamagnetic iron oxide nanoparticle (SPION) generates heat. Understanding the local heating mechanism of a SPION in suspension and in a mesoporous silica nanoparticle (MSN) will advance the design of hyperthermia-based nanotheranostics and AMF-stimulated drug delivery in biomedical applications. The AMF-induced heating of single-domain SPION can be explained by the Néel relaxation (reorientation of the magnetization) or the Brownian relaxation (motion of the particle). The latter is investigated using fluorescence depolarization based on detecting the mobility-dependent polarization anisotropy (r) of two luminescence emission bands at different wavelengths corresponded to the europium-doped luminescent SPION (EuSPION) core and the silica-based intrinsically emitting shell of the core-shell MSN. The fluorescence depolarization experiments are carried out with both the free and the silica-encapsulated SPION nanoparticles with and without application of the AMF. The r value of a EuSPION core-mesoporous silica shell in the presence of the AMF does not change, indicating that no additional rotational motion of the core-shell nanoparticles is induced by the AMF, disproving the contribution of Brownian heating and thus supporting Néel relaxation as the dominant heating mechanism.

Transient Depolarization Spectroscopic Study on Electronic Structure and Fluorescence Origin of Graphene Oxide.

It is well-established that the electronic states of graphene oxide (GO) consist of sp2 clusters with different sizes and the surrounding sp3 matrix according to recent reports. However, addressing the excitation energy migration/redistribution among those electronic states in GO-based complex systems from spectroscopic experiments is still a challenge. Here, we combine the time-resolved absorption and fluorescence depolarization experiments to reveal the excitation energy migration processes in electronic states in GO. We demonstrate that, in sp3 domains of GO, there are charge-transfer states between sp3-hybridized carbon atoms and the oxygen-containing functional groups, and the energy redistribution and charge migration in sp3 matrix occur on the time scale from subpicoseconds to tens of picoseconds. In contrast, the electronic states of sp2 clusters in GO are rather localized and dominantly contribute to the excitation-wavelength-dependent red fluorescence of GO.

Insight into the Modification of Polymeric Micellar and Liposomal Nanocarriers by Fluorescein-Labeled Lipids and Uptake-Mediating Lipopeptides.

Encapsulation of diagnostic and therapeutic compounds in transporters improves their delivery to the point of need. An even more efficient treatment of diseases can be achieved using carriers with targeting or protecting moieties. In the present work, we investigated micellar and liposomal nanocarriers modified with fluorescein, peptides, and polymers that are covalently bound to fatty acids or phospholipids to ensure a self-driven incorporation into the micelles or liposomes. First, we characterized the photophysics of the fluorescent probes in the absence and in the presence of nanocarriers. Changes in the fluorescence decay time, quantum yield, and intensity of a fluorescein-labeled fatty acid (fluorescein-labeled palmitic acid [fPA]) and a fluorescein-labeled lipopeptide (P2fA2) were found. By exploiting these changes, we investigated a lipopeptide (P2A2 as an uptake-mediating unit) in combination with different nanocarriers (micelles and liposomes) and determined the corresponding association constant Kass values, which were found to be very high. In addition, the mobility of fPA was exploited using fluorescence correlation spectroscopy (FCS) and fluorescence depolarization (FD) experiments to characterize the nanocarriers. Cellular uptake experiments with mouse brain endothelial cells provided information on the uptake behavior of liposomes modified by uptake-mediating P2A2 and revealed differences in the uptake behavior between pH-sensitive and pH-insensitive liposomes.

Identifiability analysis of rotational diffusion tensor and electronic transition moments measured in time-resolved fluorescence depolarization experiment.

The subject of this paper is studies of the deterministic identifiability of molecular parameters, such as rotational diffusion tensor components and orientation of electronic transition moments, resulting from the time-resolved fluorescence anisotropy experiment. In the most general case considered, a pair of perpendicularly polarized emissions enables the unique determination of all the rotational diffusion tensor's principal components. The influence of the tensor's symmetry and the associated degeneration of its eigenvalues on the identifiability of the electronic transitions moments is systematically investigated. The analysis reveals that independently of the rotational diffusion tensor's symmetry, the transition moments involved in photoselection and emission processes cannot be uniquely identified without a priori information about their mutual orientation or their orientation with respect to the principal axes of the tensor. Moreover, it is shown that increasing the symmetry of the rotational diffusion tensor deteriorates the degree of the transition moments identifiability. To obtain these results analytically, a novel approach to solve bilinear system of equations for Markov parameters is applied. The effect of the additional information, obtained from fluorescence measurements for different molecular mobilities, to improve the identifiability at various levels of analysis is shown. The effectiveness and reliability of the target analysis method for experimental determination of the molecular parameters is also discussed.

Theory of third-order spectroscopic methods to extract detailed molecular orientational dynamics for planar surfaces and other uniaxial systems.

Functionalized organic monolayers deposited on planar two-dimensional surfaces are important systems for studying ultrafast orientational motions and structures of interfacial molecules. Several studies have successfully observed the orientational relaxation of functionalized monolayers by fluorescence depolarization experiments and recently by polarization-resolved heterodyne detected vibrational transient grating (HDTG) experiments. In this article we provide a model-independent theory to extract orientational correlation functions unique to interfacial molecules and other uniaxial systems based on polarization-resolved resonant third-order spectroscopies, such as pump-probe spectroscopy, HDTG spectroscopy, and fluorescence depolarization experiment. It will be shown (in the small beam-crossing angle limit) that five measurements are necessary to completely characterize the monolayer's motions: I(∥)(t) and I(⊥)(t) with the incident beams normal to the surface, I(∥)(t) and I(⊥)(t) with a non-zero incident angle, and a time averaged linear dichroism measurement. Once these measurements are performed, two orientational correlation functions corresponding to in-plane and out-of-plane motions are obtained. The procedure is applicable not only for monolayers on flat surfaces, but any samples with uniaxial symmetry such as uniaxial liquid crystals and aligned planar bilayers. The theory is valid regardless of the nature of the actual molecular motions on interface. We then apply the general results to wobbling-in-a-cone model, in which molecular motions are restricted to a limited range of angles. Within the context of the model, the cone angle, the tilt of the cone relative to the surface normal, and the orientational diffusion constant can be determined. The results are extended to describe analysis of experiments where the beams are not crossing in the small angle limit.

On the analyses of fluorescence depolarisation data in the presence of electronic energy migration. Part II: Applying and evaluating two-photon excited fluorescence

Electronic energy migration within a bifluorophoric molecule has been studied by time-resolved two-photon excited (TPE) fluorescence depolarisation experiments. Data were analysed by using a recently developed quantitative approach [O. Opanasyuk and L. B.-Å. Johansson, On the Analyses of Fluorescence Depolarisation Data in the Presence of Electronic Energy Migration. Part I: Theory and General Description, Phys. Chem. Chem. Phys., submitted]. The energy migration occurs between the 9-anthrylmethyl groups of the bifluorophoric molecule, bis-(9-anthrylmethylphosphonate) bisteroid. These groups undergo local reorientations, while overall tumbling of the bisteroid is strongly hampered in the used viscous solvent, 1,2-propanediol. To solely obtain information about local reorientations of the 9-anthrylmethyl group, also the mono-(9-anthrylmethylphosphonate) bisteroid was studied, which enabled modelling of the ordering potential shape. The analysis of data is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process was described by an extended Förster theory (EFT). A reasonable value of the distance between the 9-anthrylmethyl groups, as well as for the mutual orientation of the ordering potentials, is found. Furthermore, values of the two-photon tensor components were obtained.

Activation and molecular recognition of the GPCR rhodopsin – Insights from time-resolved fluorescence depolarisation and single molecule experiments

The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.

Biophysical properties of membrane lipids of anammox bacteria: II. Impact of temperature and bacteriohopanoids

Anammox bacteria possess unique membranes that are mainly comprised of phospholipids with extraordinary “ladderane” hydrocarbon chains containing 3 to 5 linearly concatenated cyclobutane moieties that have been postulated to form relatively impermeable membranes. In a previous study, we demonstrated that purified ladderane phospholipids form fluid-like mono- and bilayers that are tightly packed and relatively rigid. Here we studied the impact of temperature and the presence of bacteriohopanoids on the lipid density and acyl chain ordering in anammox membranes using Langmuir monolayer and fluorescence depolarization experiments on total lipid extracts. We showed that anammox membrane lipids of representatives of Candidatus “Kuenenia stuttgartiensis”, Candidatus “Brocadia fulgida” and Candidatus “Scalindua” were closely packed and formed membranes with a relatively high acyl chain ordering at the temperatures at which the cells were grown. Our findings suggest that bacteriohopanoids might play a role in maintaining the membrane fluidity in anammox cells.

Excited-State Symmetry and Reorientation Dynamics of Perylenes in Liquid Solutions: Time-Resolved Fluorescence Depolarization Studies Using One- and Two-Photon Excitation

The excited-state symmetry and molecular reorientation of perylene, 1,7-diazaperylene, and 2,5,8,11-tetra- tert-butylperylene have been studied by different fluorescence depolarization experiments. The first excited electronic singlet state was reached through one-photon excitation (OPE) and two-photon excitation (TPE). A 400 and 800 nm femtosecond laser pulse was used for this purpose, and data were collected by means of the time-correlated single-photon counting technique. It is found that the rotational correlation times for each perylene derivative are very similar in the OPE and TPE depolarization experiments. For the determination of the two-photon absorption tensor, a recently described theoretical model has been applied (Ryderfors et al. J. Phys. Chem. A 2007, 111, 11531). It was found that the two-photon process can be described by a 2 x 2 absorption tensor for which the components are solvent dependent and exhibit mixed vibronic character. In the dipole approximation this is compatible with a parity-forbidden two-photon absorption into the first excited singlet state.

The Symmetry of Two-Photon Excited States as Determined by Time-Resolved Fluorescence Depolarization Experiments

A new experimental and theoretical approach is presented for the quantitative determination and assignment of the two-photon absorption tensor of fluorophores dissolved in liquid solutions. Two linearly independent time-resolved fluorescence anisotropies and the two-photon polarization ratio were determined from experiments based on using the time-correlated single photon counting technique. The data were analyzed in a global manner under the assumption of prevailing diffusive molecular reorientations and when accounting for the influence of rapid unresolved reorientations. The method has been applied in fluorescence studies of perylene, two-photon excited at 800 nm. The analysis suggests that the two-photon transition is mediated via vibronic coupling including at least two vibrations of different symmetry, and also that the first singlet excited electronic state acts as a dominating intermediate state.

Two-Photon Excited Fluorescence and Molecular Reorientations in Liquid Solutions

Theoretical expressions are derived that relate the two-photon excited fluorescence depolarisation experiments to the molecular symmetry and the rotational motions of fluorescent molecules. Diffusive rotational motions in liquid solvents are considered, as well as the influence of fast unresolved motions (e.g. librations). The results obtained are compared with one-photon excited fluorescence depolarisation experiments. The derived theoretical expressions can be applied for detailed analyses of the molecular rotation in solvent. Several of the results are useful for determining and assigning the components of two-photon absorption tensors.

Tuning Supramolecular Structuring at the Nanoscale Level: Nonstoichiometric Soluble Complexes in Dilute Mixed Solutions of Alginate and Lactose-Modified Chitosan (Chitlac)

Two oppositely charged polysaccharides, alginate and a lactose-modified chitosan (chitlac), have been used to prepare dilute binary polymer mixtures at physiological pH (7.4). Because of the negative charge on the former polysaccharide and the positive charge on the latter, polyanion-polycation complex formation occurred. A complete miscibility between the two polysaccharides was attained in the presence of both high (0.15 M) and low (0.015 M) concentrations of simple 1:1 supporting salt (NaCl), as confirmed by turbidity measurements; phase separation occurred for intermediate values of the ionic strength (I). The binary solutions were further characterized by means of light scattering, specific viscosity, and fluorescence quenching measurements. All of these techniques pointed out the fundamental role of the electrostatic interactions between the two oppositely charged polysaccharides in the formation of nonstoichiometric polyelectrolyte soluble complexes in dilute solution. Fluorescence depolarization (P) experiments showed that the alginate chain rotational mobility was impaired by the presence of the cationic polysaccharide when 0.015 M NaCl was used. Moreover, upon addition of calcium, the P values of the binary polymer mixture in 0.015 M NaCl increased more rapidly than that of an alginate solution without chitlac, suggesting an efficient crowding of the negatively charged alginate chains caused by the polycation.

On the quantitative molecular analysis of electronic energy transfer within donor–acceptor pairs

An extended Förster theory (EFT) on electronic energy transfer is presented for the quantitative analysis of time-resolved fluorescence lifetime and depolarisation experiments. The EFT, which was derived from the stochastic Liouville equation, yields microscopic information concerning the reorientation correlation times, the order parameters, as well as inter chromophoric distances. Weakly interacting donor and acceptor groups, which reorient and interact in a pair wise fashion, are considered, under isotropic and anisotropic conditions. For the analysis of experiments it is shown that not only do we need to consider the orientational distributions of the transition dipoles, but the internal reorienting molecular dynamics within the pair which is of even greater importance. The latter determines the shape as well as the rate of the observed donor fluorescence and depolarisation decays, which are most often not mono-exponential functions. It is shown that the commonly used Förster theory is a special case of the EFT. Strategies are presented for applying the EFT, which makes use of Brownian dynamics simulation.

Self-aggregation – an intrinsic property of GM1in lipid bilayers

We demonstrate that the ganglioside G(M1) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibits a non-uniform lateral distribution, i.e., enriched regions of GM(1) molecules are formed, which is an argument in favour of self-aggregation of G(M1) being an intrinsic property of G(M1) ganglioside. This was concluded from energy transfer/migration studies of BODIPY-labelled gangliosides by means of time-resolved fluorescence lifetime and depolarization experiments. Three fluorophore-labelled gangliosides were synthesized to include either of two spectroscopically different BODIPY groups. These were specifically localized either in the polar headgroup region or in the non-polar region of the lipid bilayer. An eventual ganglioside-ganglioside affinity/aggregation induced by the BODIPY groups was experimentally excluded, which suggests their use in examining the influence of G(M1) in more complex systems.

Extended Förster theory for determining intraprotein distances: 2. An accurate analysis of fluorescence depolarisation experiments

The extended Förster theory (EFT) is for the first time applied to the quantitative determination of the intramolecular distances in proteins. It is shown how the EFT (J. Chem. Phys., 1996, 105, 10896) can be adapted to the analyses of fluorescence depolarisation experiments based on the time-correlated single photon counting technique (TCSPC). The protein system studied was the latent form of plasminogen activator inhibitor type I (PAI-1), which was mutated and labelled by the thiol reactive BODIPY(R) derivative {N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide}. The energy migration occurs within pairs of photophysically identical donor groups that undergo reorientational motions on the timescales of energy migration and fluorescence relaxation. Unlike all models currently used for analysing fluorescence TCSPC data, the EFT explicitly accounts for the time-dependent reorientations that influence the rate of electronic energy transfer/migration in a complex manner. The complexity is related to the "kappa(2) problem", which has been discussed for years. The EFT brings the analyses of DDEM data to the same level of molecular description as in ESR and NMR spectroscopy, i.e. it yields microscopic information about the reorientation correlation times, the order parameters, as well as inter-chromophoric distances.

Time-Resolved Single Tryptophan Fluorescence in Photoactive Yellow Protein Monitors Changes in the Chromophore Structure during the Photocycle via Energy Transfer

We show from time-resolved fluorescence intensity and depolarization experiments that the fluorescence of the unique tryptophan W119 of PYP is quenched by energy transfer to the 4-hydroxycinnamoyl chromophore. Whereas the intensity decay has a time constant of 0.18 ns in P, the decay in the absence of the cofactor (apo-PYP) has a single exponential lifetime of 4.8 ns. This difference in lifetime with and without acceptor can be explained quantitatively on the basis of energy transfer and the high-resolution X-ray structure of P, which allows an accurate calculation of the kappa2 factor. Fluorescence depolarization experiments with donor and acceptor indicate that both are immobilized so that kappa2 is constant on the fluorescence time scale. Using background illumination from an LED emitting at 470 nm, we measured the time-resolved fluorescence in a photostationary mixture of P and the intermediates I2 and I2'. The composition of the photostationary mixture depends on pH and changes from mainly I2 at low pH to predominantly I2' at high pH. The I2/I2' equilibrium is pH-dependent with a pKa of approximately 6.3. In I2 the lifetime increases to approximately 0.82 ns. This is not due to a change in distance or to the increase in spectral overlap but is primarily a consequence of a large decrease in kappa2. Kappa2 was calculated from the available X-ray structures and decreases from approximately 2.7 in P to 0.27 in I2. This change in kappa2 is caused by the isomerization of the acceptor, which leads to a reorientation of its transition dipole moment. We have here a rare case of the kappa2 factor dominating the change in energy transfer. The fluorescence decay in the light is pH-dependent. From an SVD analysis of the light/dark difference intensity decay at a number of pH values, we identify three species with associated lifetimes: P (0.18 ns), I2 (0.82 ns), and X (0.04 ns). On the basis of the pH dependence of the amplitudes associated with I2 and X, with a pKa of approximately 6.3, we assign the third species to the signaling state I2'. The absorption spectra of the 0.82 and 0.04 ns species were calculated from the pH dependence of their fluorescence amplitudes and of the photostationary light/dark difference absorption spectra. The lambda(max) values of these spectra (372 and 352 nm) identify the 0.82 and 0.04 ns components with I2 and I2', respectively, and validate the fluorescence data analysis. The mutant E46Q allows a further test of the energy transfer explanation, since lowering the pH in the dark leads to a bleached state with an increased spectral overlap but without the isomerization-induced decrease in kappa2. The measured lifetime of 0.04 ns is in excellent agreement with predictions based on energy transfer and the X-ray structure.

Hoechst 33258 as a pH‐Sensitive Probe to Study the Interaction of Amine Oxide Surfactants with DNA

The use of Hoechst 33258 (HO) as a fluorescent probe to characterize the interactions between DNA and pH-sensitive amphiphiles is discussed. In the case of amine oxide amphiphiles dodecyldimethylamine oxide (DDAO) and p-dodecyloxybenzyldimethylamine oxide (pDoAO), the decrease in fluorescence emission, which signals DNA-amphiphile association, is accompanied by a large hypsochromic shift in the emission maximum of the bound probe; this eventually reaches a value characteristic of HO in a neutral or slightly basic environment. These findings are compared to results obtained by using the more common ethidium bromide (EB) probe, which shows no such shift. Circular dichroism and fluorescence depolarization experiments indicate that fluorescence emission only occurs from the DNA-bound probe, and the observed shift in emission maximum when using HO as a fluorescent probe is due to a variation in the local pH in the vicinity of the probe.

Preparation of Liposomes Surface-Loaded with Cationic Manganese Porphyrins as SOD Mimics

A water-soluble cationic 5,10,15,20-tetrakis(N-methylpyridinium-4- yl)porphinatomanganese(III) ion (MnT4MPyP) and water-insoluble 5,10,15,20-tetrakis(N-3-furyl)porphinatomanganese(III) ion (MnT3FuP) were prepared and ion-complexed with an anionic phospholipid membrane to enhance the circulation persistence of the porphyrin complexes in vivo. Fluorescence spectra and fluorescence depolarization experiments indicated MnT3FuP to be situated within the hydrophobic interior of the vesicle composed of the phospholipid membrane, while MnT4MPyP was anchored to the membrane by lipophilic tails with the metalloporphyrin head-group located on the interface between the membrance and aqueous solvent. SOD activity was determined by stopped-flow analysis and cytochrome c assay, which allowed the determination of IC50 and kcat values for the reaction of the metalloporphyrins with superoxide anion radical (O2-·). Sodium stearate-linked MnT4MPyP was the most effective catalyst as an SOD mimic to decompose O2-· at a second-order rate constant of 1.9×107 M-1s-1 in dimyristoylphosphatidylcholine (DMPC) liposomes. SOD activity of MnT3FuP was less than that of MnT4MPyP.

Partial donor–donor energy migration (PDDEM): A novel fluorescence method for internal protein distance measurements

We show that the photophysics of chemically identical but photophysically non-identical fluorescent pairs can be used for measuring distances within proteins. For this purpose, the theory of partial donor–donor energy migration (PDDEM, S. Kalinin, J. G. Molotkovsky and L. B.-A. Johansson, Spectrochim. Acta, Part A, 2002, 58, 1087–1097) was applied for distance measurements between BODIPY groups covalently linked to cystein residues in plasminogen activator inhibitor of type 2 (PAI-2). Two sulfhydryl specific derivatives of BODIPY were used namely; N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl) methyl iodoacetamide. To determine distances, the time-resolved fluorescence relaxation for two singly labelled forms of PAI-2, as well as the corresponding doubly labelled protein were combined and analysed in a global manner. Fluorescence depolarisation experiments on the labelled mutants were also analysed. The distances determined by PDDEM were in good agreement to those obtained from donor–donor energy migration (DDEM) experiments and structural data on PAI-2. The PDDEM approach allows for the use of very different fluorescent probes, which enables wide range of distances to be measured. The PDDEM model also provides a rational explanation to why previous observations of poly-fluorophore-labelled proteins exhibit a shorter average fluorescence lifetime compared to the arithmetic average of lifetimes obtained for the corresponding single labelled proteins.

Excimer Formation of Dimethyl 2,6-Naphthalene Dicarboxylate Embedded in a Poly(methyl methacrylate) Matrix

Dimethyl 2,6-naphthalene dicarboxylate (DMN) was embedded in a poly(methyl methacrylate) (PMMA) matrix. According to the molecular weight of the repeating unit of PMMA different samples containing 0.02 up to 8 mol % DMN have been prepared. Starting from 1 mol % DMN content, excimer formation is observed. From time-resolved fluorescence experiments the fluorescence lifetime of the monomer was derived to be 15 ns and the excimer lifetime amounts to 30 ns. It was shown that Forster excitation energy transfer takes place among the DMN monomers and the Forster radius of the monomer was determined by time-resolved fluorescence depolarization experiments to be 23 ± 3 A. Excitation energy transfer is assumed to be the dominant process for the excimer formation. Using a model of three-dimensional transport among DMN donors until an excimer trap is reached, dimensionless trap concentrations of 0.005 and 0.014 were derived for the samples with 1 and 2 mol % DMN, respectively. For larger DMN concentration the excitat...