Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.     

The New Fluorescent Membrane Probe Ahba: A Comparative Study with the Largely Used Laurdan

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.     

New Insights on the Fluorescent Emission Spectra of Prodan and Laurdan

Prodan and Laurdan are fluorescent probes largely used in biological systems. They were synthetized to be sensitive to the environment polarity, and their fluorescent emission spectrum shifts around 120 nm, from cyclohexane to water. Although accepted that their emission spectrum is composed by two emission bands, the origin of these two bands is still a matter of discussion. Here we analyze the fluorescent spectra of Prodan and Laurdan in solvents of different polarities, both by decomposing the spectrum into two Gaussian bands and by computing the Decay Associated Spectra (DAS), the latter with time resolved fluorescence. Our data show that the intensity of the lower energy emission band of Prodan and Laurdan (attributed, in the literature, to the decay of a solvent relaxed state) is higher in cyclohexane than in water, showing a decrease as the polarity of the medium increases. Moreover, in all solvents studied here, the balance between the two emission bands is not dependent on the temperature, strongly suggesting two independent excited states. Both bands were found to display a red shift as the medium polarity increases. We propose here a new interpretation for the two emission bands of Prodan and Laurdan in homogeneous solvents: they would be related to the emission of two independent states, and not to a pair of non-relaxed and solvent relaxed states.     

Membrane lipid domains and dynamics as detected by Laurdan fluorescence

2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation. In bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). TheGP value can be used for the quantitation of each phase. The wavelength dependence of theGP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined.GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in natural membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 Å.     

Membrane Dipole Potential as Measured by Ratiometric 3-Hydroxyflavone Fluorescence Probes: Accounting for Hydration Effects

We previously applied the electrochromic modulation of excited-state intramolecular proton-transfer (ESIPT) reaction for the design of novel 3-hydroxyflavone (3-HF) derivatives as fluorescent probes for measuring the dipole potential, Ψ_D, in lipid bilayers (Klymchenko et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 11219). In the present work, this method was revisited to take into account the influence of the bilayer hydration on the emission ratiometric response of 3-HF probes. For this reason, it was necessary to deconvolute the whole fluorescence spectra into three bands corresponding to the non H-bonded forms, normal N^* and tautomer T^* forms, both participating to the ESIPT reaction, and to the H-bonded H–N^* form, excluded from this reaction. This allowed us to determine the pure N^*/T^* intensity ratio, without any contribution from the H–N^* form emission depending essentially on the bilayer hydration. This new approach allowed us to confirm the correlation we obtained between the response of 3-HF probes on dipole potential modifications and the corresponding response of the reference fluorescent probe di-8-ANEPPS, thus further confirming the potency of 3-HF probes as excellent emission ratiometric probes to measure dipole potential in lipid membranes.     

Magnetic field effects on the fluorescence of Cr3+ in GdAlO3

The fluorescence spectrum of Cr³⁺ in GdAlO₃ has been examined at 4.2 K as a function of magnetic field up to 60 kG. The resulting splitting of the ²E → ⁴A₂ emission lines are explained in terms of a modified molecular field approximation, which incorporates the effect of the spin fluctuations. The exchange constant in the relaxed excited state is found to be 1.2 cm^-1, which differs from that reported from absorption data. It is suggested that the difference may be related to the Frank-Condon effect.     

Molecule formation and energy transfer processes in a vapor with high density of 3P-excited sodium atoms

A rich fluorescence spectrum extending between 4000 and 8200 Å has been observed whenever sodium vapor is excited by dye laser light tuned to the 3²S → 3²P transition. Molecule formation due to collisions between excited and unexcited atoms is manifested by the presence of an emission band of sodium in the spectral range 4160–4570 Å.     

Spectroscopy of calcium deposited on large argon clusters

Spectroscopic experiments have been performed, providing emission and excitation spectra of calcium atoms trapped on argon clusters of average size 2 000. The two experimental spectra fall in the vicinity of the calcium resonance line ¹P ₁ → ¹S₀ at 422.9 nm. The excitation spectrum consists in two bands located on each side of the resonance line of the free calcium. In addition, Monte Carlo calculations, coupled to Diatomics-In-Molecule potentials are employed to simulate the absorption spectrum of a single calcium atom in the environment of a large argon cluster of average size 300. The theoretical absorption spectrum confirms the existence of two bands, and shows that these bands are characteristic of a calcium atom located at the surface of the argon cluster and correspond to the excited 4p orbital of calcium either perpendicular or parallel to the cluster surface. The precise comparison between the shape of the absorption spectrum and that of the fluorescence excitation spectrum shows different intensity ratios. This could suggest the existence of a non adiabatic energy transfer that quenches partly the fluorescence of trapped calcium. Another explanation, although less likely, could be a substantial dependence of the calcium oscillator strength according to the alignment of the calcium excited orbital with respect to the cluster surface. The emission spectrum always shows a band in the red of the resonance line which is assigned to the emission of calcium remaining trapped on the cluster. When exciting the blue band of the excitation spectrum, the emission spectrum shows a second, weak, component that is assigned to calcium atoms ejected from the argon clusters, indicating a competition between ejection and solvation. Received 7 May 2002 Published online 1st October 2002 RID="a" ID="a"e-mail: jmm@drecam.saclay.cea.fr RID="b" ID="b"URA 2453 du CNRS RID="c" ID="c"UMR 5626 du CNRS     

Light emission by swift He+ ions after focusing and channeling interactions on a monocrystalline nickel surface

The light emission from 195 keV He⁺ ions excited in scattering at a (101) Ni surface is used to indicate anisotropy effects of beam steering at monocrystalline surfaces. Experiments and computer simulations give clear evidence of focusing by surface channels and of maximum and minimum critical angles for planar channeling.     

Tailoring of the spectral–directional characteristics of rare-earth fluorescence by metal–dielectric planar structures

It is demonstrated that the spectrum, direction and polarization of rare-earth fluorescence can be tailored by embedding the impurity ions into a planar metal–dielectric structure (MDS). The latter was designed by spin coating a rare-earth-doped oxide film (TiO₂:Sm³⁺) onto a gold-covered glass substrate. For spectral–directional investigations of Sm³⁺ fluorescence, the MDS was attached to a semi-cylindrical prism and excited by UV light from the flat side. An angular scan revealed a strongly polarized and directional emission of Sm³⁺ from the convex side of the prism. The tuning of TiO₂ film thickness in the MDS allows a control of the polarization and direction of the emission bands. A theoretical modeling of the reflectivity of the MDS suggests that the observed angular resonances in the fluorescence emission are caused by its effective coupling with surface plasmons on the gold–dielectric interface or coupling with leaky modes in sufficiently thick dielectric films working as a waveguides.     

Dual-Fluorescence Probe of Environment Basicity (Hydrogen Bond Accepting Ability) Displaying no Sensitivity to Polarity

3-Hydroxyquinolones (3HQs) are a new class of water soluble dual fluorescence probes that can monitor both polarity and basicity (H-bond accepting ability) parameters. Both parameters play an important role in proteins and lipid membranes. Nevertheless, no method exists actually to measure the basicity parameter separately from the polarity. To achieve this aim, we synthesized 2-benzofuryl-3-hydroxy-4(1H)-quinolone (3HQ-Bf) and characterized its photophysical properties by UV, steady-state and time-resolved fluorescence spectroscopy. Due to its extended conjugation and totally planar conformation, 3HQ-Bf is characterized by a high fluorescence quantum yield. In solution, this dye shows an excited state intramolecular proton transfer (ESIPT) reaction resulting in two tautomer bands in the emission spectra. The ESIPT reaction can be considered as irreversible and is governed by rate constants from 0.6 to 8 × 10⁹ s⁻¹, depending on the solvent. The analysis of the spectral properties of 3HQ-Bf in a series of organic solvents revealed a marginal sensitivity to the solvent polarity, but an exquisite sensitivity to solvent basicity, as shown by the linear dependence of the logarithm of the emission bands intensity ratio, log(I_N*/I_T*), as well as the absorption or emission maxima wavenumbers as a function of the solvent basicity parameter. This probe may find useful applications through coupling to a protein ligand, for characterizing the H-bond acceptor ability at the ligand binding site as well as for studying the basicity changes of lipid membranes during their chemo- and thermotropic conversions.     

Competition between relaxation and decay of a resonantly excited core-hole state

By a many-body theory it is shown that in general when the time scale of the relaxation from the resonantly excited core-hole state to the fully relaxed core-hole state is much shorter than that of core-hole decay, the autoionization (deexcitation) spectrum is more or less identical to the Auger-electron spectrum by core-hole decay of the fully relaxed core-hole state. Here, the fully relaxed core-hole state is the lowest core-hole state in the X-ray photoelectron spectroscopy spectrum. The present theory explains why the C 1s autoionization spectrum of CO molecule adsorbed on Ni(1 0 0) surface measured at the resonant core-level electron excitation energy is more or less indentical to the Auger-electron spectrum measured at far above the core-level electron ionization limit.     

Interctions of laurdan with membranes at high pressures

Abstract

The fluorescence emission spectra of Laurdan in egg yolk phosphatidylcholine vesicles were measured as a function of pressure. The results suggest that the long hydrocarbon side chain stabilizes Laurdan in bilayers at high pressures.     

Fluorescence Anisotropy of Tyrosinate Anion Using One-, Two- and Three-Photon Excitation

We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250–310 nm), two-photon (570–620 nm) and three-photon (750–930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pK_a 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λ_max 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565–580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at ?60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos⁶ θ to cos² θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.     

Relation Between Fluorescence Decays and Temporal Evolution of Excited States

A differential equation system describing the temporal evolution of excited substates and fluorescence emission were tested using a DOPRI algorithm. The numerical solutions show that there is significant difference in the measurable parameters according to the type of connectivity among the excited substates. In the globally connected case, the fluorescence emission exhibits a double exponential behavior, and the first moment of the emitted spectrum decays with stretched exponential characterized by β < 1. In the diffusive case the fluorescence emission cannot be always fitted with double exponential, and the first moment of the emitted spectrum may decay with stretched exponential characterized by β > 1. Details of modeling and the possibilities of drawing conclusions are also presented.     

Synthesis, characterization and fluorescence studies of a novel europium complex based sensor

A novel europium(III) complex was synthesized using TTA (α-thenoyltrifluoroacetone) as the first ligand and H₂bpdc (2,2′-bipyridine-3,3′-dicarboxylate) as the second ligand. Elemental analysis, thermal analysis, IR and UV–vis spectrum and fluorescence spectrum of the europium(III) complex were carried out. A characteristic Eu³⁺ fluorescence emission was observed in ethanol–water (1:1) solution, indicating that the complex is stable in solution and the emission of Eu(III) ions was not influenced by the water molecules. The fluorescence emission of the complex was quenched completely by the Co²⁺ and Fe³⁺ ions, but the quenched emission was recovered in the presence of glycine. Moreover, the Eu³⁺ emission was very sensitive to pH, so the complex can be used as pH-dependent fluorescence probe or chemosensors.     

Characterization of Standard Reference Material 2941, uranyl-ion-doped glass, spectral correction standard for fluorescence

Standard Reference Material^® (SRM^®) 2941 is a cuvette-shaped, uranyl-ion-doped glass, recommended for use for relative spectral correction of emission and day-to-day performance validation of fluorescence spectrometers. Properties of this standard that influence its effective use or contribute to the uncertainty in its certified emission spectrum have been explored here. These properties include its photostability, absorbance, dissolution rate in water, anisotropy, temperature coefficient of fluorescence intensity, and fluorescence lifetimes. The expanded uncertainties in the certified spectrum are about 4% around the peak maximum at 526 nm, using an excitation wavelength of 427 nm. The SRM also exhibits a strong resistance to photodegradation, with no measurable decrease in fluorescence intensity even after 8 h of laser irradiation.     

Steady-State and Time-Resolved Fluorescence Polarization Behavior of Acenaphthene

Steady-state and time-resolved fluorescence polarization studies have been carried out on acenaphthene (ACE) in low-temperature glass solutions and at room temperature. In the low-temperature glass the fluorescence polarization values vary considerably with both emission and excitation wavelength. There is a time dependence (on the nanosecond time scale) of the fluorescence anisotropy, r(t), at 77 K, which has a strong dependence upon the excitation and emission wavelengths. Under these conditions, the time-dependent decay of the anisotropy is not attributable to chromophoric motion. The observations are consistent with emission from two closely lying and interconverting excited states. Rate constants for the photophysical processes involved have been determined by fitting the data using a model proposed by Fleming et. al. The results are discussed with particular reference to the care required in using dynamic fluorescence polarization measurements to determine energy transfer rates in systems containing this chromophore.     

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r ₀) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-l-tryptophanamide.Abbreviations LADH liver alcohol dehydrogenase - -NAD⁺ -nicotinamide adenine dinucleotide - OPE one-photon excitation - OPIF one-photon induced fluorescence - TPE two-photon excitation - TCSPC time-correlated single photon counting - TPIF two-photon induced fluorescence     

Electrooptical Absorption Measurements (EOAM) Testify Existence of two Conformers of Prodan and Laurdan with Different Dipole Moments in Equilibrium Ground and Franck-Condon Excited State

The results from the electrooptical absorption measurements (EOAM) on the equilibrium ground and excited Franck-Condon state dipole moments of Prodan and Laurdan in 1,4-dioxane are presented. As follows from experiments Prodan and Laurdan in the equilibrium ground and excited Franck-Condon state have two conformers with considerably different dipole moments. The electrical dipole moments and the transition dipole moment, obtained from the short-wavelength region of the absorption spectrum are parallel. The electrical dipole moments measured at the long-wavelength spectral region are parallel to each other but not parallel to the transition dipole moment m _a. The angle θ between the transition dipole moment m _a and the dipole moment in the equilibrium ground state μ _g of the long-wavelength conformer is about 30⁰ for both probes. Obtained results evidence that donor-acceptor pairs of the short-wavelength and long-wavelength conformers are not located on the same axis. Two low-energy conformers of Prodan have been found by density functional theory (DFT) calculations, differing in the orientation of the carbonyl group towards the naphthalene system.