High-resolution silicon photonics focused ultrasound transducer with a sub-millimeter aperture.

We present an all-optical focused ultrasound transducer with a sub-millimeter aperture and demonstrate its capability for high-resolution imaging of tissue ex vivo. The transducer is composed of a wideband silicon photonics ultrasound detector and a miniature acoustic lens coated with a thin optically absorbing metallic layer used to produce laser-generated ultrasound. The demonstrated device achieves axial resolution and lateral resolutions of 12 µm and 60 µm, respectively, well below typical values achieved by conventional piezoelectric intravascular ultrasound. The size and resolution of the developed transducer may enable its use for intravascular imaging of thin fibrous cap atheroma.

Two-Dimensional Laser Melting of Lunar Regolith Simulant Using the MOONRISE Payload on a Mobile Manipulator.

Laser melting experiments were carried out with the MOONRISE payload, installed on the mobile manipulator, MIRA3D. The MOONRISE payload was developed to demonstrate the feasibility of additive processing of lunar regolith with the help of lasers on the Moon within a lunar surface mission in the next years. The development of hardware for the flight to the moon is well advanced and, if successful, would pave the way for the use of laser melting for production of components from regolith. The aim of the experiments described in this article was to test the planned scenario on the Moon, especially the interaction between laser payload, manipulator, and soil surface, and to identify suitable process parameters for production of two-dimensional (2D) objects. The ability to produce 2D objects is an important intermediate step on the way to produce large three-dimensional structures such as habitats, walls, or foundations. During the experiments, specimens with a size of ∼20 × 20 × 4 mm were repeatedly produced. As analog material, two synthetic lunar soils produced with the modular regolith simulant systems from Technische Universität Braunschweig (TUBS) were used. The experiments were conducted under Earth gravity and atmospheric conditions. This article describes the hardware used, procedure for carrying out the experiments, and properties of the produced samples.

Silicon-photonics focused ultrasound detector for minimally invasive optoacoustic imaging.

One of the main challenges in miniaturizing optoacoustic technology is the low sensitivity of sub-millimeter piezoelectric ultrasound transducers, which is often insufficient for detecting weak optoacoustic signals. Optical detectors of ultrasound can achieve significantly higher sensitivities than their piezoelectric counterparts for a given sensing area but generally lack acoustic focusing, which is essential in many minimally invasive imaging configurations. In this work, we develop a focused sub-millimeter ultrasound detector composed of a silicon-photonics optical resonator and a micro-machined acoustic lens. The acoustic lens provides acoustic focusing, which, in addition to increasing the lateral resolution, also enhances the signal. The developed detector has a wide bandwidth of 84 MHz, a focal width smaller than 50 µm, and noise-equivalent pressure of 37 mPa/Hz1/2 - an order of magnitude improvement over conventional intravascular ultrasound. We show the feasibility of the approach and the detector's imaging capabilities by performing high-resolution optoacoustic microscopy of optical phantoms with complex geometries.

Three-megadalton complex of methanogenic electron-bifurcating and CO2-fixing enzymes.

The first reaction of the methanogenic pathway from carbon dioxide (CO2) is the reduction and condensation of CO2 to formyl-methanofuran, catalyzed by formyl-methanofuran dehydrogenase (Fmd). Strongly reducing electrons for this reaction are generated by heterodisulfide reductase (Hdr) in complex with hydrogenase or formate dehydrogenase (Fdh) using a flavin-based electron-bifurcation mechanism. Here, we report enzymological and structural characterizations of Fdh-Hdr-Fmd complexes from Methanospirillum hungatei. The complexes catalyze this reaction using electrons from formate and the reduced form of the electron carrier F420. Conformational changes in HdrA mediate electron bifurcation, and polyferredoxin FmdF directly transfers electrons to the CO2 reduction site, as evidenced by methanofuran-dependent flavin-based electron bifurcation even without free ferredoxin, a diffusible electron carrier between Hdr and Fmd. Conservation of Hdr and Fmd structures suggests that this complex is common among hydrogenotrophic methanogens.

Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction.

In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

Towards artificial methanogenesis: biosynthesis of the [Fe]-hydrogenase cofactor and characterization of the semi-synthetic hydrogenase.

The greenhouse gas and energy carrier methane is produced on Earth mainly by methanogenic archaea. In the hydrogenotrophic methanogenic pathway the reduction of one CO2 to one methane molecule requires four molecules of H2 containing eight electrons. Four of the electrons from two H2 are supplied for reduction of an electron carrier F420, which is catalyzed by F420-reducing [NiFe]-hydrogenase under nickel-sufficient conditions. The same reaction is catalysed under nickel-limiting conditions by [Fe]-hydrogenase coupled with a reaction catalyzed by F420-dependent methylene tetrahydromethanopterin dehydrogenase. [Fe]-hydrogenase contains an iron-guanylylpyridinol (FeGP) cofactor for H2 activation at the active site. FeII of FeGP is coordinated to a pyridinol-nitrogen, an acyl-carbon, two CO and a cysteine-thiolate. We report here on comparative genomic analyses of biosynthetic genes of the FeGP cofactor, which are primarily located in a hmd-co-occurring (hcg) gene cluster. One of the gene products is HcgB which transfers the guanosine monophosphate (GMP) moiety from guanosine triphosphate (GTP) to a pyridinol precursor. Crystal structure analysis of HcgB from Methanococcus maripaludis and its complex with 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol confirmed the physiological guanylyltransferase reaction. Furthermore, we tested the properties of semi-synthetic [Fe]-hydrogenases using the [Fe]-hydrogenase apoenzyme from several methanogenic archaea and a mimic of the FeGP cofactor. On the basis of the enzymatic reactions involved in the methanogenic pathway, we came up with an idea how the methanogenic pathway could be simplified to develop an artificial methanogenesis system.

Millimeter-wave broadband antireflection coatings using laser ablation of subwavelength structures.

We report on the first use of laser ablation to make submillimeter, broadband, antireflection coatings (ARCs) based on subwavelength structures (SWSs) on alumina and sapphire. We used a 515 nm laser to produce pyramid-shaped structures with a pitch of about 320 µm and a total height of near 800 µm. Transmission measurements between 70 and 140 GHz are in agreement with simulations using electromagnetic propagation software. The simulations indicate that SWS-ARCs with the fabricated shape should have a fractional bandwidth response of Δν/νcenter=0.55 centered on 235 GHz for which reflections are below 3%. Extension of the bandwidth to both lower and higher frequencies, between a few tens of gigahertz and a few terahertz, should be straightforward with appropriate adjustment of laser ablation parameters.

Polarization-dependent SERS effects of laser-generated sub-100 nm antenna structures.

Sub-100 nm antenna arrays consisting of a star-like ridge or dome-like structures with needles in their centers are prepared in thin gold films on glass substrates using femtosecond laser pulses. The needles can be bent mechanically to be horizontally aligned to the substrate surface. Controlled variation of the pulse energy allows one to obtain nanostructures of different defined morphologies. These arrays of nanostructures are covered with a thin homogeneous layer of rhodamine molecules. Raman spectra using linearly polarized laser light of 632.8 nm are taken with the laser spot centered on individual nanostructures and at positions on the unstructured film. The average Raman enhancement within the laser spot focused onto a nanostructure is two orders of magnitude higher than on the unstructured film. The nanostructures with bent needles exhibit a polarization dependence of the SERS effect, i.e., typically the enhancement is larger by about a factor of two for excitation light polarized parallel to the needle direction than for the perpendicular case. The enhancement factor of the star-like ridge structures with needles is analyzed by the finite-element method, which agrees with the experiment. We show that the variation of the SERS activity of almost similar structures arises from the inherent randomness of the hot spots created in the fabrication process. Nevertheless, these antenna structures may be useful as elements in novel SERS devices as they can be accurately positioned on a device using a cheap fabrication process compatible with microfabrication technology.

Multi-focus two-photon polymerization technique based on individually controlled phase modulation.

Multi-focus two-photon polymerization with a spatial light modulator is demonstrated. The spatial light modulator generates multi-focus spots via phase modulation technique controlled by a computer generated hologram (CGH) pattern. Each focus spot can be individually addressed in position and laser intensity. The multi-focus two-photon polymerization technique allows the fabrication of complex 2-D and 3-D structures both symmetric and asymmetric. Smooth sine curved polymerized lines with amplitude of 5 microm and a period of 200 microm were obtained by fast switching of the CGH patterns.

Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

Femtosecond laser fabricated spike structures for selective control of cellular behavior.

In this study we investigate the potential of femtosecond laser generated micrometer sized spike structures as functional surfaces for selective cell controlling. The spike dimensions as well as the average spike to spike distance can be easily tuned by varying the process parameters. Moreover, negative replications in soft materials such as silicone elastomer can be produced. This allows tailoring of wetting properties of the spike structures and their negative replicas representing a reduced surface contact area. Furthermore, we investigated material effects on cellular behavior. By comparing human fibroblasts and SH-SY5Y neuroblastoma cells we found that the influence of the material was cell specific. The cells not only changed their morphology, but also the cell growth was affected. Whereas, neuroblastoma cells proliferated at the same rate on the spike structures as on the control surfaces, the proliferation of fibroblasts was reduced by the spike structures. These effects can result from the cell specific adhesion patterns as shown in this work. These findings show a possibility to design defined surface microstructures, which could control cellular behavior in a cell specific manner.

Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants.

Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 10(5) Pa of H2 at pH 7 giving rise to two distinct S1/2 electron paramagnetic resonance (EPR) signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g = 1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependence of these two [4Fe-4S] clusters suggests that they simultaneously mediate electron and proton transfer and thus could be an essential part of the proton-translocating machinery.

Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase.

Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S=1/2 [4Fe-4S]3) cluster, the EPR spectrum of which is broadened in the presence of CoM-33SH [Duin, E.C., Madadi-Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263-268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme. FEBS Lett. 538, 81-84]. These results provide indirect evidence that the disulfide binds to the iron-sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X-ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM-SeH). Se K edge extended X-ray absorption fine structure confirms a direct interaction of the Se in CoM-SeH-treated HDR with an Fe atom of the Fe-S cluster at an Fe-Se distance of 2.4A.

Influence of spatiotemporal coupling induced by an ultrashort laser pulse shaper on a focused beam profile.

4f pulse shapers have been widely used to temporally manipulate femtosecond optical pulses by spectral filtering. When the temporal waveform is manipulated with a spatial light modulator consisting of segmented pixels, the spatial profile of the output beam also varies because of diffraction at the pixel array, which is known as a spatiotemporal coupling effect. This effect produces a complicated spatio-temporal profile near the focus of the ultrashort pulses, which may affect the interpretation of experimental results obtained with shaped ultrashort pulses. We investigate the spatial intensity distribution at the focus of temporally shaped pulses through ablation experiments. The three-dimensional space-time beam profile is also numerically calculated.

Influence of coenzyme Q(10) and cerivastatin on the flow-mediated vasodilation of the brachial artery: results of the ENDOTACT study.

AIM: Endothelial dysfunction (ED) is the functional prestep in atherosclerosis. Aim of the present study was to evaluate the effects of a potent antioxidant (coenzyme Q(10), CoQ(10)) and of cerivastatin on ED of the brachial artery. METHODS AND RESULTS: Twenty-five male patients with manifest ED (flow-mediated vasodilation [FMD%]<4.5%) were included in this prospective, randomized, cross-over study. ED of the brachial artery was assessed by the use of high-resolution ultrasound. Each patient had to pass through three treatment phases ((1) single therapy with cerivastatin (C), (2) single therapy with CoQ(10), (3) combination therapy). FMD% significantly improved throughout all treatment phases ((1) 3.50+/-4.05% vs. 8.80+/-6.39%, p=0.009; (2) -0.25+/-4.0% vs. 7.06%+/-4.39%, p=0.004; (3) 3.14+/-3.54% vs. 8.82+/-5.78%, p=0.011). C led to a significant decrease of CoQ(10) plasma levels (1.23+/-0.34 vs. 0.87+/-0.39 microg/ml, p=0.004). CONCLUSION: Our results indicate a positive influence of CoQ(10) supplementation on human ED, which appears to be independent of lipid lowering. Although large-scale studies evaluating other antioxidants failed to demonstrate a positive prognostic effect, Q(10) has never been evaluated in larger trials. Experimental as well as clinical results indicate that CoQ(10) warrants further attention in atherosclerosis research.

The membrane-bound [NiFe]-hydrogenase (Ech) from Methanosarcina barkeri: unusual properties of the iron-sulphur clusters.

The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe-4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe-4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.