Study of Organized Media Using Time-Resolved Fluorescence Spectroscopy

Many photophysical processes which occur on an ultrafast time scale in ordinary liquids become significantly retarded in organized assemblies, by two to three orders of magnitude. Recently many groups have applied ultrafast laser spectroscopy and theoretical methods to elucidate this dramatic phenomenon. Although the implications of this phenomenon in biology and chemistry are not yet fully understood, it has been demonstrated that ultrafast time-resolved fluorescence spectroscopy is a very powerful tool to study the microscopic properties of the organized assemblies and that water or other liquids confined inside these assemblies are fundamentally different from the corresponding liquid in bulk.     

New quasiparticles hystons and coherent photoinduced dynamics of molecules in polar disordered media

Using methods of statistical thermodynamics, it is shown that after pulse excitation the evolution of a “polar luminescent probe—polar disordered medium” system is described by an equation of damping vibrations. This allows the conclusion that in the solvate shell of the probe molecule synchronous rotational vibrations (librations) of the molecules of the medium occur, whose damping is caused by dielectric friction. Such a collective synchronous motion is considered as a motion of a quasiparticle called a hyston. The moment of inertia J_n and mass M_h of a hyston are defined as J_h=2m² ₁a^-3Ω₀ ^-2(ε-1)/(2ε+1), M_h=J_hM_s/J_s, where m₁ is the dipole moment of the probe molecule in the excited S₁-stute; a is the Onsager radius; Ω₀ is the cyclic frequency of harmonic vibrations of the hyston; ε is the dielectric constant; M_s and J_s are the mass and moment of inertia of a molecule of the medium, respectively. The correlation function of the motion of the molecules c(t) is a solution of the equation of hyston motion. The fluorescence response s(t) in measurements with time resolution coincides with the correlation function: s(t)=c(t). The concepts concerning hystons make it possible to describe macroscopic photoinduced coherent motion that is manifested against a background of thermal motion of the medium molecules. Institute of Molecular and Atomic Physics, National Academy of Sciences of Belarus, 70, F. Skorina Ave., Minsk, 220072, Belarus. Translated from Zhurnal Prikladnei Spektroskopii, Vol 65, No. 2, pp. 176–183, March–April, 1998.     

Effects of Internal Rotations on the Time-Resolved Fluorescence in a Bichromophoric Protein System Under the Energy Transfer Interaction

Effects of internal rotations of chromophores under the energy transfer interaction in proteins on the time-resolved fluorescence were examined by numerical calculations. Expressions used for the calculations are based on the approximations that the energy transfer takes place according to Foöurster's mechanism and the rotational motions of the energy donor and acceptor along the surfaces of cones are described by a set of rotational diffusion equations. The intensity decay of the donor depended a little on the rotational diffusion coefficient of the donor in some cases, while that of the acceptor did very little. Anisotropy of the donor decayed faster as the diffusion coefficient of the donor increased. Anisotropy decay of the acceptor markedly depended not only on the mutual configuration of the pair in the protein, but also on the diffusion coefficient of the donor. The dependence of the time-resolved fluorescence on the diffusion coefficient of the acceptor was not as great as that of the donor.     

Inter- and Intramolecular Dynamics of Pyrenyl Lipids in Bilayer Membranes from Time-Resolved Fluorescence Spectroscopy

The analysis of time-resolved fluorescence of monopyrenyl phospholipid monomer and excimer emission is reviewed using a model based on the diffusion equation for bimolecular reactions in two-dimensional planar membrane bilayers. This aspect is illustrated by the analysis of a real experiment. The method can also be extended to short-range diffusion, which can be measured for dipyrenyl phospholipids in membrane bilayers. The latter aspect is illustrated by the analysis of a simulated experiment to show the experimental requirements to recover the desired parameters.     

DNA Vector Polyethyleneimine Affects Cell pH and Membrane Potential: A Time-Resolved Fluorescence Microscopy Study

Polyethyleneimine (PEI) is one of the very efficient nonviral vectors being developed and tested for artificial gene transfer into target cells. One of its serious limitations is the significant cytotoxicity of the large amounts of free PEI in the mixtures of DNA and PEI used for transfection. To further investigate the cellular effects of free PEI, we have analyzed the PEI-induced alterations of various cell parameters such as membrane heterogeneity and fluidity, cytoplasmic pH, and plasma membrane potential in a variety of cells such as Swiss 3T3 fibroblast, Chinese hamster ovary, insect cells SF9, plant cell line BY2, and Saccharomyces cerevisae. Fluorescence probes such as Nile red, SNARF-1, and cyanine dye DiSC₂(3), coupled with the technique of picosecond time-resolved fluorescence microscopy, were used in estimating the above-mentioned cell parameters. It was found that the cell membranes were largely unperturbed by PEI. However, the cytoplasmic pH showed an increase of 0.1–0.4 units when the cells were treated with PEI. The plasma membrane potential was found to be depolarized in S. cerevisae and Swiss 3T3 cells. These results suggest that the cytotoxic effects of PEI may partly originate from inhibition of regulation of cytoplasmic pH and plasma membrane potential. Further, it is proposed that the resultant cell alterations favors the transfection process.     

Different Regimes of Ultrashort Pulse Propagation in Disordered Layered Media with Resonant Loss and Gain

Different optical nanostructures containing both loss and gain components attract ever‐increasing attention as novel advanced materials and building blocks for a variety of nanophotonic and plasmonic applications. Unique tunable optical signatures of the so‐called active metamaterials support their utilization for sensing, imaging, and signal processing on micro‐ and nanoscales. However, this tunability requires flexible control over the metamaterial parameters, which could be provided by involving a set of nonlinear interactions. In this paper, a method of governing ultrashort pulses is proposed by varying the level of a population difference disorder in a random active metamaterial. This enables us to deliver three different interaction regimes: self‐induced transparency (low disorder), localization regime (moderate disorder), and light amplification (strong disorder) corresponding to strongly different light pulse speeds. Since this control could be realized via rather plain tools, like simple pump tuning, the proposed disordered medium opens a room of opportunities for designing a peculiar active component for a whole set of highly demanded optical applications.     

Fluorescence lifetime characterization of magnesium probes: Improvement of Mg2+ dynamic range and sensitivity using phase-modulation fluorometry

We measured the Mg²⁺-dependent absorption spectra, emission spectra, quantum yields, and intensity decays of most presently available fluorescent magnesium probes. The lifetimes were found to be strongly Mg²⁺ dependent for Mag-quin-1, Mag-quin-2, magnesium green, and magnesium orange and increased 2- to 10-fold upon binding of Mg²⁺. The lifetimes of Mag-fura-2, Mag-fura-5, Mag-fura red, and Mag-indo-1 were similar in the presence and absence of Mg²⁺. Detailed timeresolved measurements were carried out for Mag-quin-2 and magnesium green using phase-modulation fluorometry. Apparent dissociation constants (K _d) were determined from the steady-state and time-resolved data. Their values were compared and discussed. Mg²⁺ sensing is described using phase and modulation data measured at a single modulation frequency. Phase angle and modulation data showed the possibility of obtaining a wider Mg²⁺-sensitive range than available from intensity measurements. A significant expansion in the Mg²⁺-sensitive range was found for Mag-quin-2 using excitation wavelengths from 343 to 375 nm, where the apparentK _d from the phase angle was found to vary from 0.3 to about 100 mM. Discrimination against Ca²⁺ was also measured for Mag-quin-2 and magnesium green. Significant phototransformation and/or photode-composition, which affect the sensitivity to Mg²⁺, were observed for Mag-quin-2 and magnesium green under intense and long illumination.     

TRANES Spectra of Fluorescence Probes in Lipid Bilayer Membranes: An Assessment of Population Heterogeneity and Dynamics

Time-resolved fluorescence of eight fluorescence probes were studied in EggPC bilayer membrane vesicles. Emission wavelength dependent fluorescence decays were analyzed in a model-independent way to obtain time resolved area normalized emission spectra (TRANES). The TRANES spectra of the probes studied were classified into four types: (1) spectra that are identical at all time (one emissive species), (2) spectra that show an isoemissive point (two emissive species), (3) spectra that shift continuously with time (slow solvation dynamics or multiple species), and (4) spectra that shift for a short time and thereafter one or two emissive species are indicated. The TRANES spectra of these eight probes, except RH421, belong to the type 1, 2, or 4. The continuous shift of the TRANES spectra that was observed for the probe RH421 is attributed to multiple ground state species and not due to slow solvation dynamics.     

激光诱导荧光确定NiB自由基一个新的电子激发态：在19000-22100 cm -1的2II态

Ni针放电产生的Ni原子与B₂H₆反应产生NiB自由基,得到了在19000～22100 cm^-1的激光诱导荧光光谱. 通过对激光诱导荧光激发谱和色散谱的研究,发现了一个新的电子跃迁序列[20.77]²II-X²∑⁺,序列共包括11个振动谱带,光谱分析得到了这个新的电子激发态和基态的跃迁寿命. 在光谱常数和跃迁寿命等数据的基础上,讨论了新发现电子激发态的电子构型.     

Spectroscopic Properties and Conformational Features of Short Linear Peptides in Solution: A Fluorescence and Molecular Mechanics Investigation

The ground- and excited-state properties of two conformationally constrained hexapeptides of general formula Boc-Bin-A₁-A₂-T-A₁-A₂-OtBu, where A₁ and A₂ are -aminoisobutyric acid (Aib) or L-alanine (Ala), Bin is an optically pure, axially chiral 1,1-binaphthyl-substituted Aib, and T (Toac) is a stable nitroxide free radical-containing Ac6c analog, were investigated in methanol solution. These peptides are denoted as (R)-Bin/Toac and (S)-Bin/Toac, depending on the chirality of the binaphthyl moiety. Electronic spectra in methanol indicate the occurrence of intramolecular exciton interaction between the naphthyl moieties of Bin, and time-resolved fluorescence measurements show a biexponential decay for both peptides examined. According to infrared (IR) absorption data in the NH stretching frequency region, and to earlier X-ray diffraction results on (S)-Bin/Toac in the crystal state, both (R)-Bin/Toac and (S)-Bin/Toac populate a 3₁₀-helix in solution with opposite screw sense, the helical handedness being determined by the chirality of binaphthyl and not by that of the Ala residues in the main chain. The combination of molecular mechanics calculations with fluorescence decay data indicate that the two observed lifetimes for each peptide arise from two conformations having different interprobe distance and orientation, in which electronic energy transfer from excited Bin to Toac takes place.     

Concept for the Traceability of Fluorescence (Beads) in Flow Cytometry: Exploiting Saturation and Microscopic Single Molecule Bleaching

We have determined the fluorescence yield of stained micro beads, used for calibration purposes in flow cytometry, as function of the irradiance of the exciting laser beam. A rate equation model has been applied to derive the number of fluorescence molecules carried by each micro bead. To derive in situ photo-physical properties of the specific dye, required for the rate equation model, we discuss an approach based on flow cytometric sorting of micro beads, which have passed two laser beams with properly chosen different irradiances, and subsequent observation of single molecule bleaching employing high sensitivity microscopy. The feasibility of our approach is demonstrated presenting first results concerning saturation of fluorescence of beads in flow and single molecule bleaching by high sensitivity microscopy.     

The Effect of Zinc Ions on the Fluorescence Spectra of (Ln,Zn)of:Eu3+(Ln=Y,La and Gd) Powder Phosphors

This research note reports the fluorescence properties of Eu³⁺-doped in three sets of (Y-Zn, La-Zn and Gd-Zn) oxy fluoride powder phosphors. on excitation with a UV-source, these phosphors have displayed orangish-red color and for which the chromaticity coordinates ([Xbar], [Ybar] have been computed. the relative fluorescence intensity ratios (R) for the different measured emission transitions have been evaluated in order to examine the effect of substitution of zinc ions in the host matrix on the fluorescence behavior. Scanning electron microscopy studies have also been performed to investigate the average grain size of the synthesized phosphor material.     

Molecular dynamics study of linear and comb-like polyelectrolytes in aqueous solution: effect of Ca2+ ions

All-atom molecular dynamics simulations were employed to provide microscopic mechanism for the salt tolerance of polyelectrolytes dispersants. The conformational variation of polyelectrolytes and interactions between COO^? groups and counterions/water molecules were also studied via radius of gyration and pair correlations functions. Sodium polyacrylate (NaPA) and sodium salts of poly(acrylic acid)–poly(ethylene oxide) (NaPA–PEO) were selected as the representative linear and comb-like polyelectrolyte, respectively. The results show that Ca²⁺ ions interact with COO^? groups much stronger than Na⁺ ions and can bring ion-bridging interaction between intermolecular COO^? groups in the NaPA systems. While in the NaPA–PEO systems, the introduced PEO side chains can prevent backbone chains from ion-bridging interactions and weaken the conformational changes. The present results can help in selecting and designing new-type efficient polyelectrolyte dispersants with good salt tolerance.