Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Lanthanide Complex-Based Fluorescence Label for Time-Resolved Fluorescence Bioassay

Different from organic fluorescence dyes, fluorescent lanthanide complexes have the fluorescence properties of long fluorescence lifetime, large Stokes shift and sharp emission profile, which makes them favorable be used as the fluorescent labeling reagents for microsecond time-resolved fluorescence bioassay. Lanthanide complex-based fluorescence labels have been successfully used for highly sensitive time-resolved fluorescence immunoassay, DNA hybridization assay, cell activity assay, and bioimaging microscopy assay. Since the technique allows easy distinction of the specific fluorescence signal of the long-lived label from short-lived background noises associated with biological samples, scattering lights (Tyndall, Rayleigh and Raman scatterings) and the optical components (cuvettes, filters and lenses), the sensitivity of fluorescence bioassay has been remarkably improved. This paper summarized the recent developments of lanthanide complex-based fluorescence labels and their applications in time-resolved fluorescence bioassays mainly based on the authors’ researches and relative publications.     

Polar Plot Representation for Frequency-Domain Analysis of Fluorescence Lifetimes

We present applications of polar plots for analyzing fluorescence lifetime data acquired in the frequency domain. This graphical, analytical method is especially useful for rapid FLIM measurements. The usual method for sorting out and determining the underlying lifetime components from a complex fluorescence signal is to carry out the measurement at multiple frequencies. When it is not possible to measure at more than one frequency, such as rapid lifetime imaging, specific features of the polar plot analysis yield valuable information, and provide a diagnostic visualization of the participating fluorescent species underlying a complex lifetime distributions. Data are presented where this polar plot presentation is useful to derive valuable, unique information about the underlying component distributions. We also discuss artifacts of photolysis and how this method can also be applied to samples where each fluorescence species shows a continuous distribution of lifetimes. Polar plots of frequency-domain data are commonly used for analysis of dielectric relaxation experiments (Cole–Cole plots), which have proved to be exceptionally useful in that field for decades. We compare this analytical tool that is well developed and extensively used in dielectric relaxation and chemical kinetics to fluorescence measurements.     

Dipyrrylmetheneboron Difluorides as Labels in Two-Photon Excited Fluorometry. Part II–Nucleic Acid Hybridization Assays

Five two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF₂ labels) with fluorescence emission maximum between 530 and 590 nm, and a frequently used rhodamine label, TAMRA, were conjugated to aminomodified oligonucleotides. The performance of the labeled oligonucleotides was studied in a separation-free nucleic acid hybridization assay using ArcDia^™ TPX bioaffinity assay technology. The results show that oligonucleotide conjugates of dipyrrylmethene-BF₂ labels provide higher two-photon excited fluorescence yield and better assay sensitivity than corresponding TAMRA conjugate. The effect of conjugation on photophysical properties of the labels and performance of the labeled oligonucleotides in separation-free hybridization assay is discussed.     

Quantitative analysis of lyotropic lamellar phases SANS patterns in powder oriented samples

We have developed a detailed numerical method based on the Caillé model to fit Small Angle Neutron Scattering profiles of powder-oriented lyotropic lamellar phases. We thus obtain quantitative values for the Caillé parameter and the smectic penetration length from which we can derive the smectic compression modulus and the membrane mean bending modulus. Our method, applied to a surfactant lamellar phase system decorated by amphiphilic copolymers, provides excellent fits for any intermembrane spacing or membrane concentration over the entire q-range of the SANS experiments. We compare our fits with those obtained from the model of Nallet et al. (J. Phys. II 3, 487 (1993)), which is reviewed. Good fits are obtained with both methods for samples exhibiting “hard” smectic order (sharp Bragg peak, moderate small angle scattering). Only our procedure, however, gives good fits in the case of “soft” smectic order (smooth Bragg peak, strong small angle scattering). A quantitative criterion to discriminate between these “soft” and “hard” samples is also proposed, based on a simple analogy with smectic-A liquid crystal in contact with an undulating solid surface. This allows us to anticipate the type of thermodynamic information that can be derived from the fits.     

Liquids in equilibrium: Beyond the hypernetted chain

Density functional techniques are used to derive a charging expression for the non-uniform density of a molecular liquid. In the atomic limit the equation reduces to an exact form due to Fixman. The theory is simplified greatly via a physical approximation that accounts for three-body correlations beyond those included in the hypernetted chain (HNC) closure of the Ornstein-Zernike (OZ) equation. The radial distribution function is obtained as a special case. The theory is tested by examining the phase behavior of two fundamental complex fluids: the homopolymer blend and diblock copolymer melts. For the former it is found, contrary to HNC theory and its molecular generalizations, that a critical temperature T_c is predicted from the structure route. This T_c scales linearly with degree of polymerization N in agreement with Flory theory. The simplest form of the theory can be considered as a way to incorporate attractive interactions within a formalism that is very similar to that of the OZ or reference interaction site model (RISM). The relevance of the theory to charged liquids is also discussed.     

Statistical theory of force-induced unzipping of DNA

The unzipping transition under the influence of external force of a dsDNA molecule has been studied using the Peyrard-Bishop Hamiltonian. The critical force F_c(T) for unzipping calculated in the constant force ensemble is found to depend on the potential parameter k which measures the stiffness associated with a single strand of DNA and on D, the well depth of the on-site potential representing the strength of hydrogen bonds in a base pair. The dependence on temperature of F_c(T) is found to be (T_D - T)^1/2 (T_D being the thermal denaturation temperature) with F_c(T_D) = 0 and F_c(0) = . We used the constant extension ensemble to calculate the average force F(y) required to stretch a base pair a y distance apart. The value of F(y) needed to stretch a base pair located far away from the ends of a dsDNA molecule is found twice the value of the force needed to stretch a base pair located at one of the ends to the same distance for y 1.0 . The force F(y) in both cases is found to have a very large value for y 0.2 compared to the critical force found from the constant force ensemble to which F(y) approaches for large values of y. It is shown that the value of F(y) at the peak depends on the value of k which measures the energy barrier associated with the reduction in DNA strand rigidity as one passes from dsDNA to ssDNA and on the value of the depth of the on-site potential. The effect of defects on the position and height of the peak in the F(y) curve is investigated by replacing some of the base pairs including the one being stretched by defect base pairs. The formation and behaviour of a loop of Y shape when one of the ends base pair is stretched and a bubble of ssDNA with the shape of an eye when a base pair far from ends is stretched are investigated.     

The effect of oxygen exposure on pentacene electronic structure

We use ultraviolet photoelectron spectroscopy to investigate the effect of oxygen and air exposure on the electronic structure of pentacene single crystals and thin films. It is found that O₂ and water do not react noticeably with pentacene, whereas singlet oxygen/ozone readily oxidize the organic compound. Also, we obtain no evidence for considerable p-type doping of pentacene by O₂ at low pressure. However, oxygen exposure lowers the hole injection barrier at the interface between Au and pentacene by 0.25 eV, presumably due to a modification of the Au surface properties.     

Frustration between syn- and anticlinicity in mixtures of chiral and non-chiral tilted smectic-C-type liquid crystals

We study the effects of mixing ferroelectric and antiferroelectric liquid-crystal compounds (FLCs and AFLCs) when the former are strictly synclinic and the latter strictly anticlinic, i.e. one mixture component exhibits only SmC* and the other only SmC a* as tilted phase. Three different paths between syn- and anticlinicity were detected: transition directly between SmC* and SmC a*, transition via the SmC_β* and SmC_γ* subphases, or by “escaping” the clinicity frustration by reducing the tilt to zero, i.e. the SmA* phase is extended downwards in temperature, separating SmC* from SmC a* in the phase diagram. The most common path is the one via the subphases, demonstrating that these phases appear as a result of frustration between syn- and anticlinic and, consequently, between syn- and antipolar order. For assessing the role of chirality, we also replaced the FLC with non-chiral synclinics. With one of the AFLCs, the route via supbhases was detected even in this case, suggesting that chirality --although necessary-- does not have quite the importance that has previously been attributed to the appearance of the subphases. The path chosen in the mixture study seemed to be determined mainly by the synclinic component, the subphase induction occurring only when the SmA*-SmC* transition was second order.