Dynamic Fluorescence Anisotropy Research Articles

Coumarin 153 Dynamics in Ethylammonium Nitrate: The Effects of Dilution with Methanol

Magic angle intensity decay and dynamic fluorescence anisotropy measurements were made on the binary solvent system composed of ethylammonium nitrate ([N2,0,0,0+][NO3−], EAN) + methanol (MeOH) across the complete EAN mole fraction range (xIL = 0–1) using the neutral dipolar solute coumarin 153 (C153) at 295 K. Stokes–Einstein–Debye (SED) hydrodynamic theory was used as a model framework to assess the C153 rotational reorientation dynamics. Departure from stick SED prediction was observed (in contrast to literature reports that used cationic or anionic dyes) and indicated a significant influence of domain nanoheterogeneity on probe dynamics. Steady-state spectroscopy indicated minimal changes in spectral peak and width with mole fraction, except at xIL = 0.3 where absorption widths decreased by ~170 cm−1, signaling that C153 sensed a change in solution heterogeneity. Magic angle intensity decays corroborated the steady-state observation and the excited-state lifetimes showed a marked change from xIL = 0.2–0.4 where EAN-EAN interactions became notably more significant. C153 average rotation times (⟨τrot⟩) showed significant solvent decoupling with increased EAN. The rotational data were fit to a power law dependence, ⟨τrot⟩ ∝ (ηT)p, where p = 0.82, demonstrating the presence of dynamic heterogeneity in the EAN/MeOH solutions. With increased EAN, rotation times showed that the heterogeneity became increasingly more significant since the rotation times systematically decreased away from the hydrodynamic stick limit.

Interaction and Dynamics in a Fully Biodegradable Glucose-Containing Naturally Abundant Deep Eutectic Solvent: Temperature-Dependent Time-Resolved Fluorescence Measurements.

A new room-temperature deep eutectic solvent (DES) composed of glucose, urea, and water has been prepared and its relaxation dynamics explored via temperature-dependent time-resolved fluorescence measurements employing hydrophilic and hydrophobic solute probes. Differential scanning calorimetry measurements indicate a glass transition temperature (Tg) of ∼236 K. Measured viscosity coefficients (η) vary from ∼600 to ∼100 cP in the temperature range 318 ≤ T/K ≤ 343 and exhibit Arrhenius-type temperature dependence with an activation energy of ∼65 kJ mol-1. Interestingly, this DES forms a stable liquid at ∼300 K but is too viscous to be accurately measured by us below 318 K. Temperature-dependent dynamic fluorescence anisotropy measurements using hydrophobic and hydrophilic solutes of similar sizes reveal bi-exponential kinetics and Arrhenius-type temperature dependence for solute rotation times (⟨τr⟩) but with significantly decreased activation energies, ∼31 kJ mol-1 (hydrophobic) and ∼21 kJ mol-1 (hydrophilic). Deviation from hydrodynamics is further reflected in the strong fractional viscosity dependence of ⟨τr⟩: ⟨τr⟩ ∝ (η/T)p with p ≈ 0.3-0.5, indicating pronounced temporal heterogeneity in the relaxation dynamics. Dynamic fluorescence Stokes shift measurements (temporal resolution ∼85 ps) produce dynamic shifts of ∼500-700 cm-1, bi-exponential solvation energy relaxation with time constants in the range ∼0.2 ns and ∼4 ns, and estimated missing amplitudes of ∼65-75%. Impact of the density difference between a nonpolar solvent and this DES on the estimated missing amplitudes is explored via measuring the temperature-dependent densities and refractive indices of this DES. Lifetime measurements suggest considerable temperature dependence for the hydrophobic solute but no such dependence for the hydrophilic one. Excitation energy dependence of fluorescence emission of various solutes with widely different lifetimes indicates mild spatial heterogeneity for this DES.

The optical properties of the silver clusters and their applications in the conformational studies of human telomeric DNA

In this study, we investigated the optical properties of the silver nanoclusters (AgNCs) labeled on the 22 bases human telomeric DNA sequences (d[AG3(T2AG3)3], Hum 22AG). The steady state spectra indicate that the formation of the G-quadruplex structure will quench the fluorescence of the AgNCs. In contrast to the AgNCs labeled on the Hum 22AG, the absorption and emission spectra of the AgNCs labeled on the duplex structure are shifted due to the change of local environment and the cluster-base interactions. Moreover, the AgNCs also represent slower spectral relaxation dynamics in the duplex structure, and the result is associated with the stronger hydrogen bond networking and the higher structural rigidity of the duplex. By comparing fluorescence anisotropy dynamics of the Hum 22AG probed by different fluorescence probes, we found that labelling the AgNCs on the DNA would suppress the wobbling motion of the chromophore. The fluorescence anisotropy study also showed an increase in the structural rigidity of the duplex structure.

Dynamics of a PEG based non-ionic deep eutectic solvent: Temperature dependence

Temperature dependent dynamics of a polyethylene glycol based non-ionic deep eutectic solvent (DES) has been investigated via both dielectric relaxation spectroscopic (DRS), and time-resolved fluorescence (TRF) Stokes shift and anisotropy measurements. The composition of the DES considered is as follows: w[fCH3CONH2+(1−f)NH2CONH2]+(1−w)PEG, where w represents the weight fraction and f represents the mole fraction. In this investigation we have kept w fixed at 0.67 and f fixed at 0.6. Both DR and TRF measurements have been carried out within the temperature range, 308≤T(K)≤333. DRS measurements have been performed within the frequency window, 0.2 ≤ ν/GHz ≤ 50. TRF measurements employ coumarin 153 (C153), a fluorescent probe molecule, as a local reporter. Interestingly, the estimated static dielectric constant (ε0) for this DES is considerably large, even higher than the ε0 value of polar solvents like, dimethyl formamide and acetonitrile. Measured DR spectra require four relaxation processes, spread over sub-10 ps to nanosecond time regime, to adequately describe the detected DR dynamics of this DES. Surprisingly, this non-ionic DES possesses a nanosecond relaxation component similar to that in ionic acetamide DESs. The presence of this slow relaxation component is further supported by the corresponding dynamic fluorescence Stokes shifts measurements. Both DR and dynamic fluorescence anisotropy data reveal substantial fractional viscosity dependence for solute rotation in this DES.

Time-resolved fluorescence anisotropy study of organic lead halide perovskite

The photoluminescent polarization properties of organic halide perovskites are of great importance for the fundamental understanding and for device fabrication, while limited relevant work has been published. In this work we investigate the fluorescence polarization properties of the CH3NH3PbI3, CH3NH3PbI3−xClx, and CH3NH3PbBr3 perovskites which have been used in some of the highly efficient solar device to date. Time-resolved fluorescence anisotropy measurements were conducted on these perovskites fabricated by different methods, and with or without the capping hole transport layer. We demonstrate that CH3NH3PbBr3 perovskite exhibits isotropic fluorescence polarization while CH3NH3PbI3 and CH3NH3PbI3−xClx perovskites exhibit anisotropic fluorescence polarization. The composition influences significantly fluorescence anisotropy dynamics while fabrication has relatively weaker influence. The hole transport capping layer can accelerate the decay of the fluorescence anisotropy. The symmetry of perovskite structure is proposed to be the key factor to impact the fluorescence anisotropy.

Dynamic Solvent Control of a Reaction in Ionic Deep Eutectic Solvents: Time-Resolved Fluorescence Measurements of Reactive and Nonreactive Dynamics in (Choline Chloride + Urea) Melts.

Dynamic fluorescence anisotropy and Stokes shift measurements of [f choline chloride + (1 - f) urea)] deep eutectic solvents at f = 0.33 and 0.40 have been carried out using a dipolar solute, coumarin 153 (C153), in the temperature range 298 ≤ T ≤ 333 K. Subsequently, measured time-dependent solvent response is utilized to investigate the dynamic solvent control on the measured rates of photoexcited intramolecular charge transfer (ICT) reactions of two molecules, 4-(1-azetidinyl)benzonitrile (P4C) and 4-(1-pyrrolidinyl)benzonitrile (P5C), occurring in these media. Measured average reaction time scales (⟨τ(rxn)⟩) exhibit the following dependence on average solvation times scales (⟨τ(s)⟩): ⟨τ(rxn)⟩ ∝ ⟨τ(s)⟩(α) with α = 0.5 and 0.35 for P4C and P5C, respectively. Such a strong dynamic solvent control of ⟨τ(rxn)⟩, particularly for P4C, is different from earlier observations with these ICT molecules in conventional molecular solvents. Excitation wavelength-dependent fluorescence emissions of C153 and trans-2-[4-(dimethylamino)styryl]-benzothiazole (DMASBT), which differ widely in average fluorescence lifetimes (⟨τ(life)⟩), suggest the presence of substantial spatial heterogeneity in these systems. Dynamic heterogeneity is reflected via the following fractional viscosity (η) dependences of ⟨τ(s)⟩ and ⟨τ(r)⟩ (⟨τ(r)⟩ being solute's average rotation time): ⟨τx⟩ ∝ (η/T)(p) with 0.7 ≤ p ≤ 0.9. Different correlations between ⟨τ(s)⟩ and ⟨τ(r)⟩ emerge at different temperature regimes, indicating variable frictional coupling at low and high temperatures. Estimated dynamic Stokes shifts in these media vary between ∼1200 and ∼1600 cm(-1), more than 50% of which possess a time scale much faster than the temporal resolution (∼75 ps) employed in these measurements. Estimated activation energy for η is closer to that for ⟨τ(r)⟩ than that for ⟨τ(s)⟩, suggesting ⟨τ(s)⟩ being more decoupled from η than ⟨τ(r)⟩.

Probing the Microenvironment in the Confined Pores of Mesoporous Silica Nanoparticles.

The microenvironment inside of the pores of mesoporous silica nanoparticles is probed using spectroscopic techniques. The probe molecules are sealed inside of the pores by a nanovalve system that is capable of controlling the access to the pore and ensuring the exclusive probing of the pore environment without any interference from the probe molecules on the outer surface of the particles or from the surrounding solution. Rigidochromism studies are used to evaluate the rigidity of the solvent matrix inside of the pore, and dynamic fluorescence anisotropy experiments are employed to determine the rotational diffusion freedom of the probe molecule. The data show that those probe molecules are neither completely free to move nor tightly attached to the pore wall, and their mobility is changed by altering the charge of the pore wall.

The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer

A simple and sensitive fluorescence anisotropy method was developed for lysozyme, employing the coupling of fluorophore, 6-carboxyfluorescein (FAM), with lysozyme upon recognition between the target molecule and its DNA aptamer. It was found in this study that the rotational dynamic of the detecting system is crucial to obtain a high anisotropy signal that cannot always be achieved by simply increasing the molecular volume, because molecular volume increase may not be able to efficiently retard the rotational movement of the fluorophore. FAM was selected as the label of the ssDNA aptamer to effectively facilitate the change of the fluorophore from a primarily independent segmental movement to slow global rotation. The time-resolved measurements, including lifetime and dynamic fluorescence anisotropy, were conducted to study the recognition interaction and to better understand the methodology. The proposed method had a wide linear dynamic range of 12.5–300 nM and a high sensitivity with the limit of detection of 4.9 nM (3S/N). This proposed method was successfully applied to assay of human salivary lysozyme. The results based on the standard addition recovery and comparison with enzyme-linked immunosorbent assay (ELISA) demonstrated the feasibility of this method for biological samples. Using coupling between the fluorophore and the analyte can be one of the approaches working toward expanding the application of fluorescence anisotropy based on aptamer–target and antibody–antigen recognitions.

Global Analysis of Dynamic Fluorescence Anisotropy by a Polarized Phasor Approach

Recently, the graphical analysis of the fluorescence lifetime imaging using the phasor approach has been highlight, and a series of the reports have made it on the way for the applications by the nonprofessionals. In this paper, we put forward a similar theory validated by the experiments for the dynamic fluorescence anisotropy imaging. By subtracting the perpendicular component from the parallel one in the frequency-domain polarization measurement, we deduce a new analytical expression about the fluorescence joint time, and find that as much as the fluorophore is a single exponential decay and r∞ is equal to zero, △I(t) is a single exponential decay with the time constant X as well, and the center of its histograms is located on the semicircle in the polarized phasor plot. In the end, we conclude that the fluorescence joint time is the best parameter to weigh the fluorescence dynamics for the macromolecules.

Imaging lifetime and anisotropy spectra in the frequency domain

We report the development of a system combining the capabilities of fluorescence imaging spectroscopy (x, lambda, I), fluorescence lifetime (tau) and static and dynamic fluorescence anisotropy (r), enabling the wide-field measurement of the spectroscopic parameters of fluorophores: (x, lambda, I, tau, r). The system employs a frequency domain data collection strategy with a modulated light emitting diode as the light source. A polarization rotator placed in the excitation path after a polarizer allows alternating parallel and perpendicular images to be collected without moving parts. A second polarizer on the emission side serves as the analyzer, leading to estimations of the wavelength-dependent dynamic anisotropies. The spectrograph has a nominal range of 365-920 nm; however, the light-emitting diodes and filter sets used in this study restricted the usable range from about 510 to 700 nm. The system was tested on rhodamine 6G (R6G) solutions containing 0, 15, 37, 45, 59, 74 and 91 glycerol. These experiments gave rotational diffusion results comparing favourably with literature values while also demonstrating a trend towards shorter measured lifetimes at high refractive index. The ability of the system to resolve mixtures was tested on mixtures of anti-human IgG-FITC (gamma-chain-specific) and R6G. These fluorophores have similar lifetimes but could be separated using anisotropy parameters. The imaging capabilities of the system were tested on mixtures of fluorescent beads with glycerol solutions of R6G.

Mechanism of interaction of PITPα with membranes: Conformational changes in the C-terminus associated with membrane binding

Eukaryotic phosphatidylinositol transfer proteins (PITPs) are composed predominantly of small (∼32 kDa) soluble proteins that bind and transfer a single phospholipid, normally phosphatidylinositol or phosphatidycholine. Two forms, PITPα and PITPβ, which share approximately 80% amino acid sequence similarity, are known. Rat PITPα was labeled at specific single reactive Cys residues with I-AEDANS and used to examine PITP–membrane interactions. Upon binding to phospholipid vesicles, PITP labeled with AEDANS at the C-terminus, a region postulated to be involved in membrane binding, shows significant decreases in both steady-state and dynamic fluorescence anisotropy. In contrast, PITPs labeled with AEDANS at sites located distal to the C-terminus show increases in both steady-state and dynamic anisotropy. These results suggest that interaction of PITP with membrane surfaces leads to significant alterations in conformation and perhaps melting of the C-terminal helix.

Time-Resolved Fluorescence Investigation of Energy Transfer in Compact Phenylacetylene Dendrimers

Excitation energy transfer dynamics in light-harvesting dendritic macromolecules has generated a great deal of interest due to the fact that such systems can be used in photovoltaic and light-emitting devices. Studies on phenylacetylene-based dendrimers played a key role in stimulating this interest and extensive theoretical as well as experimental investigations have been carried out to get a better insight into the phenomena behind the excitation energy transfer dynamics of these dendrimers. In this manuscript, time-resolved femtosecond fluorescence and fluorescence anisotropy dynamics as well as temperature dependent steady state spectral studies of a second generation homogeneous (compact) phenylacetylene dendrimer are reported. The low-temperature fluorescence spectrum of the dendrimer showed two distinctive emission peaks that can be related to the existence of two closely spaced electronic states of the chromophore. Time-resolved fluorescence results suggest delocalization of the excitation energy in the excited state. The ultrafast fluorescence anisotropy decay dynamics further supports the existence of a delocalized state.

Femtosecond Fluorescence Anisotropy Kinetics as a Signature of Ultrafast Electronic Energy Transfer in Bichromophoric Molecules

Ultrafast time resolved fluorescence anisotropy decay measurements were performed to gain insight into the energy gap dependence of donor-acceptor inter-chromophoric coupling within one supra-molecule. Three new compounds, each consisting of two semi-rigidly linked and strongly coupled chromophores, were designed and synthesized for this study. Their general structure is donor-spacer-acceptor, where "donor" is phenylamino, pyrenylamino, or benzanthronylamino moiety, and acceptor is aminobenzanthrone. While being similar structurally, the compounds differ significantly in the excitation energy difference of the two chromophores in a supra-molecule. Experimental data show an ultrafast initial fluorescence emission anisotropy decrease (within less then 1 ps) when the excited state energies of the interacting chromophores are close to each other or equal. No such fast fluorescence anisotropy dynamics is observed for a compound with a large energy gap.

Time-Resolved Total Internal Reflection Fluorometry Study on Chemical and Structural Characteristics at Water/Oil Interfaces

Abstract Structures and characteristics of water/oil interfaces at the molecular level are reviewed. Dynamic fluorescence anisotropy of Sulforhodamine 101 (SR101) and excitation energy transfer from SR101 to Acid Blue 1 (AB1) at water/oil interfaces were studied by using time-resolved total internal reflection (TIR) fluorometry. The results indicated that a water/carbon tetrachloride (CCl4) interface was sharp with respect to the molecular size of SR101 (∼10 Å), while a water/1,2-dichloroethane (DCE) interface was relatively rough compared to the water/CCl4 interface. The present results were also compared with those predicted from molecular dynamic simulations and the thermally capillary wave theory. Furthermore, on the basis of fluorescence dynamic measurements of Sulforhodamine B (SRB) adsorbed at a water/oil interface, a relationship between thickness/roughness and the polarity at the interface was discussed.

Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK 27GFP and TK 366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK 366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 ± 1.6°, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 Å. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK 366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.

Restrained Torsional Dynamics of Nuclear DNA in Living Proliferative Mammalian Cells

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.

Excitation Energy Transfer from Sulforhodamine 101 to Acid Blue 1 at a Liquid/Liquid Interface: Experimental Approach To Estimate Interfacial Roughness

Dynamic fluorescence anisotropy of Sulforhodamine 101 (SR101) and excitation energy transfer from SR101 to Acid Blue 1 (AB1) at water/carbon tetrachloride (CCl4) and water/1,2-dichloroethane (DCE) interfaces were studied by using time-resolved total-internal-reflection (TIR) fluorometry. A magic-angle dependence of the TIR fluorescence dynamic anisotropy revealed that rotational reorientation of SR101 at the water/CCl4 interface was restricted in the two-dimensional plane of the interface, while that at the water/DCE interface took place rather freely, similar to reorientation in an isotropic medium. Furthermore, the structural dimension derived from the excitation energy transfer dynamics measurements at the water/CCl4 or water/DCE interface was 1.93 or 2.48, respectively. These results indicated that the water/CCl4 interface was sharp with respect to the molecular size of SR101 (∼10 A), while the water/DCE interface was relatively rough compared to the water/CCl4 interface. Structural dimension analysis...

Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement.

Fundamental aspects of the application of fluorescence anisotropy to detect the hybridization of fluorescein-labeled DNA oligomers were explored. The oligomers included a binding site for the EcoRI restriction enzyme, which binds to double-stranded DNA and is used in this work to enhance the difference between the anisotropies of the single-stranded and double-stranded oligomers by increasing the effective volume of the latter. The fluorescence anisotropy increases upon hybridization and further upon binding of EcoRI to the double strand. By varying the length of the tether used to attach the fluorescein to the 5' end of the oligonucleotide, it was found that a 6-carbon tether was optimal, providing the most dramatic increases in anisotropy in the presence of EcoRI. Dynamic fluorescence anisotropy (DFA) provided insight into the increases in steady-state anisotropy. In most cases, the best fits to the DFA data were obtained using a biexponential decay model, which describes an anisotropic rotator. Upon hybridization, the faster rotational motion is more hindered, and the contribution of the slower rotational component is increased. This effect is enhanced by binding of EcoRI to the double strand, especially when the EcoRI binding site is near the fluorescein at the 5' end and the tether length is in the optimal range. Because the rotational correlation time of the slower anisotropy decay component is much longer than the fluorescence lifetime, it is possible in some cases to reduce the anisotropic rotator model to the special limiting case of a hindered rotator.

A new approach to polydispersity studies of sodium taurocholate and sodium taurodeoxycholate aggregates using dynamic fluorescence anisotropy

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA new approach to polydispersity studies of sodium taurocholate and sodium taurodeoxycholate aggregates using dynamic fluorescence anisotropyGuang Li and Linda B. McGownCite this: J. Phys. Chem. 1993, 97, 25, 6745–6752Publication Date (Print):June 1, 1993Publication History Published online1 May 2002Published inissue 1 June 1993https://doi.org/10.1021/j100127a028RIGHTS & PERMISSIONSArticle Views170Altmetric-Citations37LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (992 KB) Get e-Alerts

Comparison of Steady-State and Dynamic Fluorescence Anisotropy of Dansyl Probe in the Volume Phase Transition of Poly(acrylamide) Gel

Comparison of Steady-State and Dynamic Fluorescence Anisotropy of Dansyl Probe in the Volume Phase Transition of Poly(acrylamide) Gel