Characterization of bonding modes in metal complexes through electron density cross-sections

Qualitative inspection of molecular orbitals (MOs) remains one of the most popular analysis tools used to describe the electronic structure and bonding properties of transition metal complexes. In symmetric coordination complexes, the use of group theory and the symmetry-adapted linear combination (SALC) of fragment orbitals allows for a very accurate and informative interpretation of MOs, but the same procedure cannot be performed for asymmetric complexes, such as Schrock and Fischer carbenes. In this work, we present a straight-forward approach for classifying and quantifying MO contributions to a particular metal–ligand interaction. Our approach utilizes the topology of MO density contributions to a cross-section of an inter-nuclear region, and is computationally inexpensive and applicable to symmetric and asymmetric complexes alike. We also apply the same approach with similar decompositions using Natural Bond Orbitals (NBO) and the recently developed Fragment, Atomic, Localized, Delocalized and Interatomic (FALDI) density decomposition scheme. In particular, FALDI analysis provides additional insights regarding the multi-centric nature of metal-carbene bonds without resorting to expensive multi-reference calculations.     

Evaluation of the rate of abiotic degradation of biodegradable polyethylene in various environments

The rate of abiotic degradation of polyethylene (PE) films containing a manganese pro-degradant has been studied in various environments at 60 and 70 °C. The degradation was monitored from the change in molecular weight and the elongation at break after exposure to dry and humid air. It was observed that moisture had a strong accelerating effect on the rate of thermo-oxidation of PE films. However, despite the humidity level in the compost environment being similar to that in humid air, the rate of degradation in compost was much slower. It is proposed that ammonia and/or hydrogen peroxide generated by microorganisms in the compost can be responsible for the deactivating effect, as aqueous solutions of these compounds significantly retard the rate of degradation.     

Ratiometric FRET-based detection of DNA and micro-RNA in solution

A ratiometric method for detecting DNA oligomers in bulk solution based on Förster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).     

LIGHT QUENCHING OF FLUORESCENCE: A NEW METHOD TO CONTROL THE EXCITED STATE LIFETIME AND ORIENTATION OF FLUOROPHORES

Abstract Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modem high-repetition rate lasers, a phenomenon we call “light quenching.” In this overview article, we describe the possible effects of light quenching on the steady-state and time-resolved intensity and anisotropy of fluorophores. One can imagine two classes of experiments. Light quenching can occur within the single excitation pulse, or light quenching can be accomplished with a second time-delayed quenching pulse. The extent of light quenching depends on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Depending upon the polarization of the light quenching beam, light quenching can decrease or increase the anisotropy. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The polarization can increase to unity under selected conditions. Quenching with time-delayed light pulses can result in step changes in the intensity or anisotropy, which is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. These predicted effects of light quenching, including oscillations in the frequency-domain data, were demonstrated to occur using selected fluorophores. The increasing availability and use of pulsed laser sources requires consideration of the possible effects of light quenching and offers the opportunity for a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent quenching pulses to modify the excited state population, followed by time- or frequency-domain measurement of the optically prepared excited fluorophores.     

Plastic Versus Glass Support for an Immunoassay on Metal-Coated Surfaces in Optically Dense Samples Utilizing Directional Surface Plasmon-Coupled Emission

We compared plastic (polycarbonate) and high-quality glass support materials for gold-coated slides, when performing a model immunoassay against rabbit IgG using fluorescently labeled (AlexaFluor-647) anti-rabbit IgG, and detecting surface plasmon-coupled emission (SPCE) signals. Both, glass and plastic slides were simultaneously coated with a 48-nm layer of gold and protected with a 10-nm layer of silica. The maximum SPCE signal of AlexaFluor-647 was only two- to three-fold smaller on plastic slides than on glass slides. A small difference in the SPCE angles on glass (θ _F = 55°) and plastic (θ _F = 52.5°) slides was observed and can be explained with a slightly smaller refractive index of the plastic. We have not found any difference in the angle distribution (sharpness of the fluorescence signal at optimal SPCE angle) for the plastic slide compared to the glass slide. The kinetics of binding was monitored on the plastic slide as well as on the glass slide. Optically dense samples, a 4% red blood cell suspension and a 15% hemoglobin solution, are causing a reduction in the immunoassay SPCE signal by approximately 15% and three times, respectively, and the percentage of the reduction is the same for plastic and for glass slides. We believe that plastic substrates can be readily used in any SPCE assay, with only marginally lower total signal compared to high-quality glass slides.     

Microcomputer-controlled insturmentation for voltammetric techniques

A simple computer system for conducting voltammetric experiments is described. An Atari 800 XL microcomputer is connected to a laboratory-made analog/digital voltammetric analyzer by means of a two-directional serial transmission interface. The serial transmission makes it possible to decrease the number of connectors, which allows optical coupling between the microcomputer and other parts of the system. The system provides the user with computer-aided execution of experiments, including synthesis of potential waveforms and collection and development of responses. The performance of the hardware system is demostrated by its application in determining cadmium at the ng 1^-1 level in a solution of Suprapur sulphuric acid by background-corrected anodic stripping voltammetry with a deposition time of 180 s and a scan rate of 9 V s^-1.     

Photostability of Cy3 and Cy5-Labeled DNA in the Presence of Metallic Silver Particles

We examined the photostability of a double-stranded DNA oligomer, covalently labeled with Cy3 or Cy5 on one strand, in the presence of metallic silver island films. In our experimental configuration a minor fraction of the labeled DNA was close to the silver particles and the remainder was distant from the particles. Proximity of the fluorophores to silver island films resulted in increased intensity. Upon continuous illumination we found a fraction of the emission that was resistant to the photobleaching. The emission spectra of the residual fractions were identical to the initial spectra. The frequency-domain lifetime measurements of this fraction revealed greatly shortened decay times. These results are consistent with the photostable fraction being close to the silver particles. This results suggest that the number of photons detected per fluorophore, prior to photobleaching, can be increased 5-fold or more by proximity to silver particles. Localization at an optimal distance from the silver surface may result in larger enhancements.     

Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10^–6, 0.44×10^–6, and 4.3×10^–6 cm²/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.Dedicated to Professor Alfons Kawski on the occasion of his 65th birthday.     

CALCIUM-DEPENDENT FLUORESCENCE LIFETIMES OF INDO-1 FOR ONE- AND TWO-PHOTON EXCITATION OF FLUORESCENCE

Abstract— We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca²⁺. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in thc multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 × 10⁻⁴⁹ cm⁴ s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-photon excitation, using either emission wavelength ratios or lifetime imaging methods.     

Picosescond fluorescence lifetime standards for frequency- and time-domain fluorescence

We characterized a series of dimethylamino-stilbene derivatives as standards for time-domain and frequency-domain lifetime measurements. The substances have reasonable quantum yields, are soluble in solvents available with a high purity, and do not show significant sensitivity to oxygen quenching. All the fluorophores displayed single exponential intensity decays, as characterized by frequency-domain measurements to 10 GHz. The decay times vary from 880 to 57 ps, depending on structure, solvent, and temperature, which is a useful range for modern picosecond time-domain or gigahertz frequency-domain instruments. These fluorophores may be used either to test an instrument or as reference compounds to eliminate color effects. We also characterized two-fluorophore mixtures, with the decay times spaced twofold (150 and 300 ps), with varying proportions. These mixtures are useful for testing the resolution of other time- and frequency-domain instrumentation. The excitation wavelength ranges from 260 to 430 nm, and the emission from 350 to 550 nm. The decay times are independent of the excitation and emission wavelengths.