INSTRUMENTATION FOR FLUORESCENCE MICROSCOPY WITH PICOSECOND TIME RESOLUTION

Abstract— Time-resolved fluorescence microscopy using excitation by actively mode-locked dye lasers and analysis by time-correlated single photon counting is shown to be an effective way of obtaining a high degree of spatial and temporal resolution. The imaging capabilities of the microscope make for optimal instrument response functions even with inexpensive photomultiplier tubes. Thus far limited (by the laser source) to long wavelength visible excitation, the excellent light collection and imaging, coupled with the sensitivity of single photon counting make it highly probable that the much weaker U-V second harmonics of the visible dyes will be useable. Certainly the potential of using the third harmonic line (355 nm) of a mode-locked c.w. Nd: YAG laser, or fundamental lines from mode-locked c.w. ion lasers as excitation sources will enhance the technique. Nevertheless, with visible-absorbing dyes only it is possible to excite such fluorochromes as chlorophylls, porphyrins, xanthenes (rose bengal, erythrosin B), phycobiliproteins, thionine dyes, ethidium bromide, and so on. Furthermore, this technique can be straightforwardly extended for polarized light measurements thereby allowing determinations of rotational diffusion of fluorochromes in cells and organelles. The extension to variable temperature situations is easy to conceive. In addition to its use for examination of cellular and sub-cellular entities, the equipment described can be profitably employed wherever spatial resolution may provide extra information, such as studies of powders and the structures of surfaces and interfaces.     

The reaction of 2-picoline N-Oxide with o-substituled benzoates and lsatoic anhydrides

The reaction of 2-picoline N-Oxide anion with o-substituted henzoates and isatoic anhydrides to give synthetically useful l-(2-methoxyphenyl)-2-(2-pyridinyl)ethanone N-oxides, I-(2-hydroxy-phenyJ)-2-(2-pyridinyl)ethanone N-oxides and I -(2-arninopheny])-2-(2-pyridinyl)ethanone N-oxides is described.     

Femtosecond study of Cu(H(2)O) dynamics

The short-time nuclear dynamics of Cu(H(2)O) is investigated using femtosecond photodetachment-photoionization spectroscopy and time-dependent quantum wave packet calculations. The Cu(H(2)O) dynamics is initiated in the electronic ground state of the complex by electron photodetachment from the Cu(-)(H(2)O) complex, where hydrogen atoms are oriented toward Cu. Several time-resolved resonant multiphoton ionization schemes are used to probe the ensuing reorientation and dissociation. Immediately following photodetachment, the neutral complex is far from its minimum energy geometry and possesses an internal energy comparable to the Cu-H(2)O dissociation energy and undergoes both large-amplitude H(2)O motion and dissociation. Dissociation is observed to occur on three distinct time scales: 0.6, 8, and 100 ps. These results are compared to the results of time-dependent J=0 wave packet calculations, propagating the initial anion vibrational wave functions on the ground-state potential of the neutral complex. An excellent agreement is obtained between the experimental results and the ionization signals derived from the calculated probability amplitudes. Related experiments and calculations are carried out on the Cu(D(2)O) complex, with results very similar to those of Cu(H(2)O).     

Complex emulsion inversion pattern associated with the partitioning of nonionic surfactant mixtures in the presence of alcohol cosurfactant

Commercial ethoxylated nonionic surfactant mixtures containing alcohol cosurfactant exhibit a three-phase behavior whose formulation strongly varies with the water/oil ratio. As a consequence, a change in water/oil ratio can result in a sequence of up to three different emulsion inversion processes, through a combination of formulation and composition effects.     

Wittig and horner-wittig coupling reactions of 2-substituted cyclic ethers and their application to spiroketal synthesis

Wittig and Horner-Wittig coupling reactions of tetrahydropyran or tetrahydrofuran 2-triphenylphosphonium salts or 2-diphenylphosphine oxides with aldehydes and lactols affords good yields of the corresponding enol ethers. In selected examples these enol ether products may be further converted to spiroketals some of which are natural pheromones derived from $\text{Dacus oleae}$ and $\text{Paravespula vulgaris}$.     

Origin of enantioselection in chiral alcohol oxidation catalyzed by Pd[(-)-sparteine]Cl2

A kinetic investigation into the origin of enantioselectivity for the Pd[(-)-sparteine]Cl(2)-catalyzed aerobic oxidative kinetic resolution (OKR) is reported. A mechanism to account for a newly discovered chloride dissociation from Pd[(-)-sparteine]Cl(2) prior to alcohol binding is proposed. The mechanism includes (1) chloride dissociation from Pd[(-)-sparteine]Cl(2) to form cationic Pd(-)-sparteine]Cl, (2) alcohol binding, (3) deprotonation of Pd-bound alcohol to form a Pd-alkoxide, and (4) beta-hydride elimination of Pd-alkoxide to form ketone product and a Pd-hydride. Utilizing the addition of (-)-sparteine HCl to control the [Cl(-)] and [H(+)] and the resulting derived rate law, the key microscopic kinetic and thermodynamic constants were extracted for each enantiomer of sec-phenethyl alcohol. These constants allow for the successful simulation of the oxidation rate in the presence of exogenous (-)-sparteine HCl. A rate law for oxidation of the racemic alcohol was derived that allows for the successful prediction of the experimentally measured k(rel) values when using the extracted constants. Besides a factor of 10 difference between the relative rates of beta-hydride elimination for the enantiomers, the main enhancement in enantiodetermination results from a concentration effect of (-)-sparteine HCl and the relative rates of reprotonation of the diastereomeric Pd-alkoxides.     

The synthesis and thermal curing parameters of a novel quinoxaline monomer with terminal ethynyl thermoset and phenylethynyl intramolecular cycloaddition postcure capability

The Synthesis of 3,3′-bis(4-[3-ethynylphenoxy]phenyl)-7,7′-bis(phenylethynyl)-2,2′-diphenyl-6,6′-biquinoxaline (I) was accomplished by the reaction of 2,2′-bis(phenylethynyl)-5,5′-diaminobenzidine (II) and 4-(3-ethynylphenoxy)benzil. Thermal analysis of I indicated a softening temperature of 107°C, followed by an exotherm above 150°C that corresponded to a independent crosslinking reaction of the terminal acetylene groups and an intramolecular cycloaddition (IMC) reaction of the 2,2′-bis(phenylethynyl)biphenyl moieties. In the synthetic work substantial improvements were made in the synthesis of II. The sample of I was cured at 200°C and the maximum partially cured transition temperature attained was 280°C. A sample of 3,3′-bis(4,[3-ethynylphenoxy]phenyl)-2,2′-diphenyl-6,6′-biquinoxaline (IV) was similarly tested as a model without IMC capability and its corresponding value was 250°C. The difference between these two values is discussed briefly.     

Reactivity of DNA guanyl radicals with phenolate anions

Guanine bases are the most easily oxidized sites in DNA. Electron-deficient guanine species are major intermediates produced in DNA by the direct effect of ionizing radiation (ionization of the DNA itself) because of preferential hole migration within DNA to guanine bases. By using thiocyanate ions to modify the indirect effect (ionization of the solvent), we are able to produce these single-electron-oxidized guanine radical species in dilute aqueous solutions of plasmid DNA where the direct effect is negligible. The guanyl radical species produce stable modified guanine products. They can be detected in the plasmid by converting them to strand breaks after incubation with a DNA repair enzyme. If a phenol is present during irradiation, the yield of modified guanines is decreased. The mechanism is reduction of the guanine radical species by the phenol. It is possible to derive a rate constant for the reaction of the phenol with the guanyl radical. The pH dependence shows that phenolate anions are more reactive than their conjugate acids, although the difference for guanyl radicals is smaller than with other single-electron-oxidizing agents. At physiological pH values, the reduction of a guanyl radical entails the transfer of a proton in addition to the electron. The relatively small dependence of the rate constant on the driving force implies that the electron cannot be transferred before the proton. These results emphasize the potential importance of acidic tyrosine residues and the intimate involvement of protons in DNA repair.     

Nanoparticle-cored dendrimers: synthesis and characterization

The synthesis and characterization of a group of new dendrimers-namely, nanoparticle-cored dendrimers (NCDs)-are described. These materials were obtained by the reduction of hydrogen tetrachloroaurate phase-transferred into toluene in the presence of Fréchet-type polyaryl ether dendritic disulfide wedges of generation 1-5. These materials, possessing nanometer-sized gold clusters at the core and dendritic wedges radially connected to the core by Au-S bonds, were analyzed by TEM and TGA, and by UV, IR, and NMR spectroscopies. The number of branching units connected to the core decreased with the generation of the dendritic wedge, and this number changed from 2.18/nm(2) for Au-G-2 to 0.27/nm(2) for Au-G-5. This result suggests that, in the higher-generation NCDs, a large fraction of the surface area of the metal cluster is not passivated and is therefore available for catalytic activity.     

The anionic alkylation of easily reducible arenes. A photochemical route to nucleophilic aromatic substitution of anthracene by organolithiums

High yield nucleophilic addition ensues upon mixing tetrahydrofuran solutions of benzyllithium or cyclooctadienyllithium and anthracene at low temperatures At least part of this addition proceeds by a single electron transfer pathway. Photolysis of the resulting adducts leads to the elimination of lithium hydride, giving net nucleophilic substitution in approximately 50% yield. The analogous reaction fails with naphthalene, where photolysis of the organolithium/ arene mixture leads to dimeric products derived from the organolithium.