Real‐Time Nanomicroscopy via Three‐Dimensional Single‐Particle Tracking

We developed a new method for real‐time, three‐dimensional tracking of fluorescent particles. The instrument is based on a laser‐scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual‐color, wide‐field setup. Dual‐color fluorescence wide‐field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent‐labeled artificial viruses in tubulin‐eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resolution.     

A new photostable terrylene diimide dye for applications in single molecule studies and membrane labeling

A new terrylene diimide-based dye (WS-TDI) that is soluble in water has been synthesized, and its photophysical properties are characterized. WS-TDI forms nonfluorescing H-aggregates in water that show absorption bands being blue-shifted with respect to those of the fluorescing monomeric form. The ratio of monomeric WS-TDI to aggregated WS-TDI was determined to be 1 in 14 400 from fluorescence correlation spectroscopy (FCS) measurements, suggesting the presence of a large amount of soluble, nonfluorescent aggregates in water. The presence of a surfactant such as Pluronic P123 or CTAB leads to the disruption of the aggregates due to the formation of monomers in micelles. This is accompanied by a strong increase in fluorescence. A single molecule study of WS-TDI in polymeric films of PVA and PMMA reveals excellent photostability with respect to photobleaching, far above the photostability of other common water-soluble dyes, such as oxazine-1, sulforhodamine-B, and a water-soluble perylenediimide derivative. Furthermore, labeling of a single protein such as avidin is demonstrated by FCS and single molecule photostability measurements. The high tendency of WS-TDI to form nonfluorescent aggregates in water in connection with its high affinity to lipophilic environments is used for the fluorescence labeling of lipid membranes and membrane containing compartments such as artificial liposomes or endosomes in living HeLa cells. The superior fluorescence imaging quality of WS-TDI in such applications is demonstrated in comparison to other well-known membrane staining dyes such as Alexa647 conjugated with dextran and FM 4-64 lipophilic styryl dye.     

Characterization of self-assembled supramolecular [Ga4L6] host-guest complexes by electrospray ionization mass spectrometry

Self-assembled supramolecular host-guest complexes have been characterized by electrospray ionization mass spectrometry. The spectra obtained by use of a Q-TOF instrument equipped with a Z-spray ion source show primarily the 3- and 4- charge states of the assemblies. The assemblies have the general formula [guest subset Ga4L6]11- where L represents the chelating bidentate catechol ligand 1,5-bis(2',3'-dihydroxy-benzamido)naphthalene and guests are tetramethyl ammonium (Me4N+), tetraethyl ammonium (Et4N+), tetra-n-propyl ammonium (Pr4N+) and decamethylcobaltocenium (Cp*2Co+) cations. For the first time, the mass spectrum of the empty assembly [Ga4L6]12- is reported. This article also reports that provided the electrospray ion source is capable of preserving noncovalent interactions, it is possible to observe host-guest complexes containing both weak binding guests as well as sterically demanding guests in the mass spectra. The present data suggest that electrospray mass spectrometry is a powerful tool for characterization of supramolecular host-guest complexes.     

Binary inorganic salt mixtures as high conductivity liquid electrolytes for >100 degrees C fuel cells

We report the successful application of low-melting inorganic salts with protonated cations (e.g. ammonium) as electrolytes in fuel cells operating in the temperature range 100-200 degrees C, where even with unoptimized electrodes, cell performance is comparable to that of the phosphoric acid fuel cell operating with optimized electrodes in the same temperature range, while open circuit voltages, and efficiencies at low current densities, can be much better--and there is no need for humidification or pressure to sustain performance.     

Efficient alkene hydrosilation with bis(8-quinolyl)phosphine (NPN) nickel catalysts. The dominant role of silyl-over hydrido-nickel catalytic intermediates

A cationic nickel complex of the bis(8-quinolyl)(3,5-di-tert-butylphenoxy)phosphine (NPN) ligand, [(NPN)NiCl]⁺, is a precursor to efficient catalysts for the hydrosilation of alkenes with a variety of hydrosilanes under mild conditions and low catalyst loadings. DFT studies reveal the presence of two coupled catalytic cycles based on [(NPN)NiH]⁺ and [(NPN)NiSiR₃]⁺ active species, with the latter being more efficient for producing the product. The preferred silyl-based catalysis is not due to a more facile insertion of alkene into the Ni–Si (vs. Ni–H) bond, but by consistent and efficient conversions of the hydride to the silyl complex.

A cationic nickel complex of the bis(8-quinolyl)(3,5-di-tert-butylphenoxy)phosphine (NPN) ligand, [(NPN)NiCl]⁺, is a precursor to efficient catalysts for the hydrosilation of alkenes with hydrosilanes under mild conditions and low catalyst loadings.     

Amphipols can support the activity of a membrane enzyme

Amphipathic polymers ("amphipols") were introduced several years ago (Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047-15050) as an alternative method for solubilizing integral membrane proteins in stable, nativelike conformations. However, direct maintenance of full membrane protein functionality in amphipol solutions has not previously been demonstrated in the absence of added lipid or detergent. In this contribution, the first zwitterionic amphipol "PMAL-B-100" is introduced. PMAL-B-100 not only maintains membrane protein structure and solubility, but also supports the full catalytic activity of an integral membrane enzyme, diacylglycerol kinase, in the complete absence of additional lipid or detergent. All of the roles which a lipid bilayer normally plays in maintaining diacylglycerol kinase's structure and in facilitating catalysis are satisfied by the environment and interactions supplied by PMAL-B-100.     

Elimination of methane from protonated acetaldehyde: The Ab initio transition state

The transition state (TS) for loss of CH₄ from protonated acetaldehyde has been located at the second-order Moller-Plesset (MP2)/6-31G(d,p) level of theory. The activation energy is predicted to be 263.9 kJ/mol starting from the more stable form (methyl and hydrogen E) and 261.6 kJ/mol starting from the less stable form (methyl and hydrogen Z) that is required for reaction. The products (methane and the formyl ion) are predicted to lie 136.6 kJ/mol below the TS for their formation. MP2 methods underestimate the heats of formation of both the TS and the reaction products by about 40 kJ/mol when compared with experiment. Restricted Hartree-Fock (RHF) calculations give much more accurate relative energies. The MP2 TS leads directly to fragmentation and is described as a protonation of the methyl group by the acidic proton on oxygen. Under RHF theory the reaction is stepwise. An RHF TS similar to the MP2 TS leads to a nonclassical intermediate (which is stable at this level of theory) that has one of the C---H bonds protonated. This mechanism (protonation of an alkyl group) appears to be a general one for high energy 1,2 eliminations from organic cations. (J Am Soc Mass Spectrom 1994, 5, 1102-1106)     

Investigation of the degradation of commercial polyoxymethylene copolymer in water service applications

An investigation has been carried out on the influence of degradation on various properties of commercial polyoxymethylene (POM) copolymer fittings in chlorinated cold water service lines. Scanning electron microscope (SEM) was used to document interior bulk morphological changes and typical defects. The various types of irregular cracks and pits were observed at the innerface of the samples. DSC and intrinsic viscosity determination were carried out for investigation of crystallinity and molecular weight changes. It was seen that the crystallinity of the POM increases at a much higher rate at the inner surface layers than the outer layer in the used samples. The FT-IR spectra of the new and used samples indicate the extent of degradation of the samples.