Isolation of the effect of the hairy layer length on the mechanical properties of waterborne coatings

The effect of the acidic hairy layer length on the interdiffusion of polymer between particles and as a consequence on the mechanical properties of the films produced from waterborne coatings has been studied. In order to isolate this effect, latexes with the same particle diameter and molecular weight but stabilized with poly(acrylic acid)-block-poly(butyl acrylate) (PAA-b-PBA) block copolymers of controlled and different lengths were prepared. Tensile strength measurements showed at the macroscopic level that the presence of AA chains in the particle surface reduced the mechanical properties of the films dried at room temperature, being its effect worse the longer the AA chain length. Higher annealing temperatures erased the negative effect of the acidic hairy layer on mechanical properties. The neutralization with NaOH instead of with NH₄OH also led to worse mechanical properties. These macroscopic results were supported by Fluorescence Resonance Energy Transfer (FRET) experiments that showed that at the microscopic level, the extent of interdiffusion occurred slower when the AA chains in the particles surface increased, the annealing temperature was lower and when NaOH was used as neutralizing agent instead of NH₄OH.     

New inhibitors of the Tat-TAR RNA interaction found with a "fuzzy" pharmacophore model

TAR RNA is a potential target for AIDS therapy. Ligand-based virtual screening was performed to retrieve novel scaffolds for RNA-binding molecules capable of inhibiting the Tat-TAR interaction, which is essential for HIV replication. We used a "fuzzy" pharmacophore approach (SQUID) and an alignment-free pharmacophore method (CATS3D) to carry out virtual screening of a vendor database of small molecules and to perform "scaffold-hopping". A small subset of 19 candidate molecules were experimentally tested for TAR RNA binding in a fluorescence resonance energy transfer (FRET) assay. Both methods retrieved molecules that exhibited activities comparable to those of the reference molecules acetylpromazine and chlorpromazine, with the best molecule showing ten times better binding behavior (IC50 = 46 microM). The hits had molecular scaffolds different from those of the reference molecules.     

Fluorescence lifetime imaging of coral fluorescent proteins

Corals, like many other coelenterates, contain fluorescent pigments that show considerable homology with the well known green fluorescent protein of the jellyfish Aequoria. In corals, unlike jellyfish, multiple proteins are present and the range of excitations and emissions suggest the possibility of energy transfer. The occurrence of F?rster resonant energy transfer (FRET) between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504, and amilFP593 (names indicate emission peaks) were 2.8, 2.9, and 2.9 ns, respectively. In the coral sample, imaging the entire emission spectrum from 420 nm, the mean lifetime was reduced to 1.5 ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3 ns, supporting this interpretation. In contrast, no reduction in lifetime is seen in the coral Euphyllia ancora, where the pigment distribution also suggests that the pigments are unlikely to be involved in photoprotection. This study set out to determine the extent of FRET between pigments in two corals, Acropora millepora and Euphyllia, ancora which differ in the arrangement of their pigments and hence possibly in pigment function.     

Correlated fluorescence lifetime and spectral measurements in living cells

Studies of proteins' interaction in cells by FRET can take benefit from two important photo-physical properties describing fluorescent proteins: fluorescence emission spectrum and fluorescence lifetime. These properties provide specific and complementary information about the tagged proteins and their environment. However, none of them taken individually can completely quantify the involved fluorophore characteristics due to their multiparametric dependency with molecular environment, experimental conditions, and interpretation complexity. A solution to get a better understanding of the biological process implied at the cellular level is to combine the spectral and temporal fluorescence data acquired simultaneously at every cell region under investigation. We present the SLiM-SPRC160, an original temporal/spectral acquisition system for simultaneous lifetime measurements in 16 spectral channels directly attached to the descanned port of a confocal microscope with two-photon excitation. It features improved light throughput, enabling low-level excitation and minimum invasivity in living cells studies. To guarantee a fairly good level of accuracy and reproducibility in the measurements of fluorescence lifetime and spectra on living cells, we propose a rigorous protocol for running experiments with this new equipment that preserves cell viability. The usefulness of SLiM approach for the precise determination of overlapping fluorophores is illustrated with the study of known solutions of rhodamine. Then, we describe reliable FRET experiments in imaging mode realized in living cells using this protocol. We also demonstrate the benefit of localized fluorescence spectrum-lifetime acquisitions for the dynamic study of fluorescent proteins. proteins.     

Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells

Fluorescent protein-based FRET is a powerful method for visualizing protein-protein interactions and biochemical reactions in living cells. It can be difficult, however, to avoid photobleaching when observing fluorescent cells under the microscope, especially those expressing CFP. We compared the sensitivity of two protein-based FRET pairs to light-induced fluorescence changes in the donor, on FRET determination by fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low excitation light levels of the time- and space-correlated single photon counting (TSCSPC) method, FLIM acquisitions were achieved without donor photobleaching. Here, we show that photobleaching of CFP by a mercury lamp under the microscope induced a decrease in the mean fluorescence lifetime, which interfered with FRET determination between CFP and YFP. Importantly, the range of light-induced variation of the mean fluorescence lifetime of CFP was not proportional to the decrease in the steady state fluorescence intensity and varied from cell to cell. The choice of the CFP/YFP pair therefore requires that the cells be observed and analyzed at very low light levels during the whole FRET experiment. In contrast, the GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if some GFP photobleaching took place. We thus demonstrate that CFP can be an unreliable donor for FRET determination in living cells, due to its photosensitivity properties. We demonstrate that the GFP/mCherry pair is better suited for FRET measurement by FLIM in living cells than the CFP/YFP pair.     

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal‐to‐noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal‐to‐noise ratio or optimize a newly defined ‘figure of merit’, which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.     

Bioinspired study of energy and electron transfer in photovoltaic system

ABSTRACT

This study focuses on understanding the fundamentals of energy transfer and electron transport in photovoltaic devices with uniquely designed nanostructures by analysing energy transfer in purple photosynthetic bacteria using dye-sensitised solar cell systems. Förster resonance energy transfer between the xanthene dye (donor of energy) and a new polymethine dye (acceptor of energy) was studied in dye-sensitised solar cells, which leads to a doubling of energy conversion efficiency in comparison to the cell with only the polymethine dye. The electron transport in the two different nanostructures of zinc oxide (nanorods and nanosheets) was investigated by spectroscopic methods (UV-vis spectrometer, time-resolved photoluminescence spectroscopy) and electrochemical potentiostat methods. The nanosheet structure of zinc oxide showed high short circuit current and long diffusion length. This fundamental study will lead to efficient artificial photosystem designs.     

Proposal of a New Method for Measuring F?rster Resonance Energy Transfer (FRET) Rapidly,Quantitatively and Non-Destructively

The process of radiationless energy transfer from a chromophore in an excited electronic state (the “donor”) to another chromophore (an “acceptor”), in which the energy released by the donor effects an electronic transition, is known as “Förster Resonance Energy Transfer” (FRET). The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle ½ and frequencies f₁ and f₂ by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at f₁, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at f₂. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at f₁ but also at the beat frequency Δf: = |f₁ − f₂|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.     

Quantitative Analysis of Tau-Microtubule Interaction Using FRET

The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αβ-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and β-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau.