Tepidiforma bonchosmolovskayae gen. nov., sp. nov., a moderately thermophilic Chloroflexi bacterium from a Chukotka hot spring (Arctic, Russia), representing a novel class, Tepidiformia, which includes the previously uncultivated lineage OLB14.

A novel aerobic moderately thermophilic bacterium, strain 3753OT, was isolated from a Chukotka hot spring (Arctic, Russia) using the newly developed technology of laser engineering of microbial systems. Сells were regular short rods, 0.4×0.8-2.0 µm in size, with a monoderm-type envelope and a single flagellum. The temperature and pH ranges for growth were 42-60 °C and pH 6.5-8.5, the optima being 50-54 °C and pH 7.3. Strain 3753OT grew chemoorganoheterotrophically on a number of carbohydrates or peptidic substrates and volatile fatty acids, and chemolithoautotrophically with siderite (FeCO3) as the electron donor. The major cellular fatty acid was branched C19 : 0. Phosphatidylethanolamine, phosphatidylglycerol and two unidentified phospholipids as well as two yellow carotenoid-type pigments were detected in the polar lipid extract. Strain 3753OT was inhibited by chloramphenicol, polymyxin B, vancomycin, streptomycin, neomycin and kanamycin, but resistant to the action of novobiocin and ampicillin. The DNA G+C content was 69.9 mol%. The 16S rRNA gene as well as 51 conservative protein sequence-based phylogenetic analyses placed strain 3753OT within the previously uncultivated lineage OLB14 in the phylum Chloroflexi. Taking into account the phylogenetic position as well as phenotypic properties of the novel isolate, the novel genus and species Tepidiforma bonchosmolovskayae gen. nov., sp. nov., within the Tepidiformaceae fam. nov., the Tepidiformales ord. nov. and the Tepidiformia classis nov. are proposed. The type strain of Tepidiforma bonchosmolovskayae is 3753OT (=VKM B-3389T=KTCT 72284T).

Cell Type-Specific Adhesion and Migration on Laser-Structured Opaque Surfaces.

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell-implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material.

High-speed two-photon polymerization 3D printing with a microchip laser at its fundamental wavelength.

High-resolution, high-speed 3D printing by two-photon polymerization (2PP) with a Nd:YVO4 Q-switched microchip laser at its fundamental wavelength of 1064 nm is demonstrated. Polymerization scan speeds of up to 20 mm/s and feature sizes of 250 nm are achieved using a high repetition rate Q-switched microchip laser with a semiconductor saturable absorber mirror (SESAM) and photoresist with a new photo-initiator bearing 6-dialkylaminobenzufuran as electron donor and indene-1,3-dione moiety as electron acceptor. The obtained results demonstrate the high potential of Q-switched microchip lasers for applications in 2PP 3D printing.

Multifocal diffractive lens generating several fixed foci at different design wavelengths.

We propose a method for designing multifocal diffractive lenses generating prescribed sets of foci with fixed positions at several different wavelengths. The method is based on minimizing the difference between the complex amplitudes of the beams generated by the lens microrelief at the design wavelengths, and the functions of the complex transmission of multifocal lenses calculated for these wavelengths. As an example, a zone plate generating three fixed foci at three different wavelengths was designed, fabricated, and experimentally investigated. The proof-of-concept experimental results confirm the formation of foci with fixed positions at the design wavelengths. The obtained results may find applications in the design and fabrication of novel multifocal contact and intraocular lenses with reduced chromatic effects.

Direct Amplitude-Phase Near-Field Observation of Higher-Order Anapole States.

Anapole states associated with the resonant suppression of electric-dipole scattering exhibit minimized extinction and maximized storage of electromagnetic energy inside a particle. Using numerical simulations, optical extinction spectroscopy, and amplitude-phase near-field mapping of silicon dielectric disks, we demonstrate high-order anapole states in the near-infrared wavelength range (900-1700 nm). We develop the procedure for unambiguously identifying anapole states by monitoring the normal component of the electric near-field and experimentally detect the first two anapole states as verified by far-field extinction spectroscopy and confirmed with the numerical simulations. We demonstrate that higher-order anapole states possess stronger energy concentration and narrower resonances, a remarkable feature that is advantageous for their applications in metasurfaces and nanophotonics components, such as nonlinear higher-harmonic generators and nanoscale lasers.

Efficient Second-Harmonic Generation in Nanocrystalline Silicon Nanoparticles.

Recent trends to employ high-index dielectric particles in nanophotonics are motivated by their reduced dissipative losses and large resonant enhancement of nonlinear effects at the nanoscale. Because silicon is a centrosymmetric material, the studies of nonlinear optical properties of silicon nanoparticles have been targeting primarily the third-harmonic generation effects. Here we demonstrate, both experimentally and theoretically, that resonantly excited nanocrystalline silicon nanoparticles fabricated by an optimized laser printing technique can exhibit strong second-harmonic generation (SHG) effects. We attribute an unexpectedly high yield of the nonlinear conversion to a nanocrystalline structure of nanoparticles supporting the Mie resonances. The demonstrated efficient SHG at green light from a single silicon nanoparticle is 2 orders of magnitude higher than that from unstructured silicon films. This efficiency is significantly higher than that of many plasmonic nanostructures and small silicon nanoparticles in the visible range, and it can be useful for a design of nonlinear nanoantennas and silicon-based integrated light sources.

Development of a biodegradable flow resisting polymer membrane for a novel glaucoma microstent.

Within this paper we analyzed the technical feasibility of a novel microstent for glaucoma therapy. For lowering of intraocular pressure, the flexible polyurethane (PUR) implant is designed to drain aqueous humour from the anterior chamber of the eye into subconjunctival, or alternatively suprachoroidal, space. The microstent includes a biodegradable, flow resisting polymer membrane serving as temporary flow resistance for the prevention of early postoperative hypotony. A biodegradable local drug delivery (LDD)-device was designed to prevent fibrous encapsulation. Biodegradable components were made of flexible, nonwoven membranes of Poly(4-hydroxybutyrate) (P(4HB)). Polymer samples and microstent prototypes were manufactured by means of dip coating, electrospinning and femtosecond-laser micromachining and characterized in vitro with regard to structural and fluid mechanical properties, degradation behavior and drug release. Bending stiffness of PUR-tubing (62.53 ± 7.57 mN mm2) is comparable to conventional glaucoma drainage devices in a tube-plate design. Microstent prototypes yield a flow resistance of 2.4 ± 0.6 mmHg/µl min-1 which is close to the aspired value corresponding to physiological pressure (15 mmHg) and aqueous humour flow (2 µl min-1) conditions inside the eye. Degradation of electrospun P(4HB) specimens was found to be almost completely finished after six months in vitro. Within this time frame, flow capacity of the microstent increases, which is beneficial to compensate potentially increasing flow resistance of fibrous tissue in vivo. Fast drug release of the LDD-device was found. One microstent prototype was implanted into a porcine eye ex vivo. Future preclinical studies will allow further information about Microstent performance.

Ultrafast surface plasmon-polariton logic gates and half-adder.

In this paper, we present a plasmonic model system for the realization of ultrafast all-optical NOT, AND, OR, and XOR gate operations using linear interference effects in dielectric crossed waveguide structures. The waveguides for the surface plasmon-polaritons are produced by a simple but highly accurate microscopic lithographic process and are optimized for single mode operation at an excitation laser wavelength of 800 nm. The functionality of the presented structures is demonstrated using sub-30 fs laser pulses from a mode locked titanium:sapphire laser. Using leakage radiation microscopy we show ultrafast SPP switching and logic operations of one basic structure consisting of two crossed waveguides with an additional output waveguide along the bisecting line of the input waveguides. The individual gates are realized on a footprint of 10 µm × 20 µm. Experimental investigations are supported by finite-difference time-domain simulations, where good agreement between experimental results and numerical simulations is obtained. To exploit the high precision of the fabrication method and its huge potential for realizing functional complex plasmonic circuitry we experimentally demonstrate a half-adder structure and its operation by combining and cascading several plasmonic waveguide components and logic gate elements on an area of only 10 µm × 28 µm.

The interplay between localized and propagating plasmonic excitations tracked in space and time.

In this work, the mutual coupling and coherent interaction of propagating and localized surface plasmons within a model-type plasmonic assembly is experimentally demonstrated, imaged, and analyzed. Using interferometric time-resolved photoemission electron microscopy the interplay between ultrashort surface plasmon polariton wave packets and plasmonic nanoantennas is monitored on subfemtosecond time scales. The data reveal real-time insights into dispersion and localization of electromagnetic fields as governed by the elementary modes determining the functionality of plasmonic operation units.

Hyaluronic acid based materials for scaffolding via two-photon polymerization.

Hydrogels are able to mimic the basic three-dimensional (3D) biological, chemical, and mechanical properties of native tissues. Since hyaluronic acid (HA) is a chief component of human extracellular matrix (ECM), it represents an extremely attractive starting material for the fabrication of scaffolds for tissue engineering. Due to poor mechanical properties of hydrogels, structure fabrication of this material class remains a major challenge. Two-photon polymerization (2PP) is a promising technique for biomedical applications, which allows the fabrication of complex 3D microstructures by moving the laser focus in the volume of a photosensitive material. Chemical modification of hyaluronan allows application of the 2PP technique to this natural material and, thus, precise fabrication of 3D hydrogel constructs. To create materials with tailor-made mechanochemical properties, HA was combined and covalently cross-linked with poly(ethylene glycol) diacrylate (PEGDA) in situ. 2PP was applied for the fabrication of well elaborated 3D HA and HA-PEGDA microstructures. For enhanced biological adaption, HA was functionalized with human epidermal growth factor.

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.

The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro.

Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions-which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance.

Spatiotemporal characterization of SPP pulse propagation in two-dimensional plasmonic focusing devices.

The spatiotemporal evolution of a SPP wave packet with femtosecond duration is experimentally investigated in two different plasmonic focusing structures. A two-dimensional reconstruction of the plasmonic field in space and time is possible by the numerical analysis of interferometric time-resolved photoemission electron microscopy data. We show that the time-integrated and time-resolved view onto the wave packet dynamics allow one to characterize and compare the capabilities of two-dimensional components for use in plasmonic devices operating with ultrafast pulses.

3D fabrication of all-polymer conductive microstructures by two photon polymerization.

A technique to fabricate electrically conductive all-polymer 3D microstructures is reported. Superior conductivity, high spatial resolution and three-dimensionality are achieved by successive application of two-photon polymerization and in situ oxidative polymerization to a bi-component formulation, containing a photosensitive host matrix and an intrinsically conductive polymer precursor. By using polyethylene glycol diacrylate (PEG-DA) and 3,4-ethylenedioxythiophene (EDOT), the conductivity of 0.04 S/cm is reached, which is the highest value for the two-photon polymerized all-polymer microstructures to date. The measured electrical conductivity dependency on the EDOT concentration indicates percolation phenomenon and a three-dimensional nature of the conductive pathways. Tunable conductivity, biocompatibility, and environmental stability are the characteristics offered by PEG-DA/EDOT blends which can be employed in biomedicine, MEMS, microfluidics, and sensorics.

Nanostripe length dependence of plasmon-induced material deformations.

Following the impact of a single femtosecond light pulse on nickel nanostripes, material deformations-or "nanobumps"-are created. We have studied the dependence of these nanobumps on the length of nanostripes and verified the link with plasmons. More specifically, local electric currents can melt the nanostructures in the hotspots, where hydrodynamic processes give rise to nanobumps. This process is further confirmed by independently simulating local magnetic fields, since these are produced by the same local electric currents.

A new perfluoropolyether-based hydrophobic and chemically resistant photoresist structured by two-photon polymerization.

Two-photon polymerization technology has been used to fabricate submicrometer three-dimensional (3D) structures using a new polyfunctional perfluoropolyether-based resist, which is a polymer intrinsically hydrophobic and chemically resistant. The fluorinated resist was designed and synthesized in this work and successfully employed to fabricate woodpile structures in various experimental conditions. This is the first demonstration of the capability to fabricate hydrophobic and chemically resistant 3D structures with submicrometer resolution and arbitrary geometry.

Urokinase receptor associates with myocardin to control vascular smooth muscle cells phenotype in vascular disease.

OBJECTIVE: The urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) are a potent multifunctional system involved in vascular remodeling. The goal of the study was to unravel the mechanisms of uPA/uPAR-directed vascular smooth muscle cell (VSMC) differentiation. METHODS AND RESULTS: Using cultured human primary VSMCs, we identified a new molecular mechanism controlling phenotypic modulation in vitro and in vivo. We found that the urokinase-type plasminogen activator receptor (uPAR) acts together with the transcriptional coactivator myocardin to regulate the VSMC phenotype. uPAR, a glycosylphosphatidylinositol-anchored cell-surface receptor family member, undergoes ligand-induced internalization and nuclear transport in VSMCs. Platelet-derived growth factor receptor ß and SUMOylated RanGAP1 mediate this trafficking. Nuclear uPAR associates with myocardin, which is then recruited from the promoters of serum response factor target genes and undergoes proteasomal degradation. This chain of events initiates the synthetic VSMC phenotype. Using mouse carotid artery ligation model, we show that this mechanism contributes to adverse vascular remodeling after injury in vivo. We then cultured cells on a microstructured biomaterial and found that substrate topography induced uPAR-mediated VSMC differentiation. CONCLUSIONS: These findings reveal the transcriptional activity of uPAR, controlling the differentiation of VSMCs in a vascular disease model. They also suggest a new role for uPAR as a therapeutic target and as a marker for VSMC phenotyping on prosthetic biomaterials.

Demonstration of magnetic dipole resonances of dielectric nanospheres in the visible region.

Strong resonant light scattering by individual spherical Si nanoparticles is experimentally demonstrated, revealing pronounced resonances associated with the excitation of magnetic and electric modes in these nanoparticles. It is shown that the low-frequency resonance corresponds to the magnetic dipole excitation. Due to high permittivity, the magnetic dipole resonance is observed in the visible spectral range for Si nanoparticles with diameters of ∼200 nm, thereby opening a way to the realization of isotropic optical metamaterials with strong magnetic responses in the visible region.

344Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity.

Generation of human neuronal networks by three-dimensional (3D) bioprinting is promising for drug testing and hopefully will allow for the understanding of cellular mechanisms in brain tissue. The application of neural cells derived from human induced-pluripotent stem cells (hiPSCs) is an obvious choice, since hiPSCs provide access to cells unlimited in number and cell types that could be generated by differentiation. The questions in this regard include which neuronal differentiation stage is optimal for printing of such networks, and to what extent the addition of other cell types, especially astrocytes, supports network formation. These aspects are the focus of the present study, in which we applied a laser-based bioprinting technique and compared hiPSC-derived neural stem cells (NSCs) with neuronal differentiated NSCs, with and without the inclusion of co-printed astrocytes. In this study, we investigated in detail the effects of cell types, printed droplet size, and duration of differentiation before and after printing on viability, as well as proliferation, stemness, differentiation potential, formation of dendritic extensions and synapses, and functionality of the generated neuronal networks. We found a significant dependence of cell viability after dissociation on differentiation stage, but no impact of the printing process. Moreover, we observed a dependence of the abundance of neuronal dendrites on droplet size, a marked difference between printed cells and normal cell culture in terms of further differentiation of the cells, especially differentiation into astrocytes, as well as neuronal network formation and activity. Notably, there was a clear effect of admixed astrocytes on NSCs but not on neurons.

Low-frequency magnetic response of gold nanoparticles.

Gold nanoparticles (AuNPs) exposed to low frequency magnetic fields have shown promise in enhancing biological processes, such as cellular reprogramming. Despite the experimental evidence, a comprehensive understanding of the underlying physical principles and the corresponding theory remains elusive. The most common hypothesis is that functionalized nanoparticles transiently amplify magnetic fields, leading to improved cellular reprogramming efficiency. However, a detailed investigation on this topic is lacking. This paper bridges this knowledge gap by conducting a comprehensive investigation on the magnetic response of surface-modified AuNPs exposed to magnetic fields with frequencies up to hundreds of MHz. Starting with the inherent properties of bulk gold material, we explore a wide range of magnetic susceptibilities that might result from the redistribution of charge carriers due to bond molecules on the particle surfaces. Through analytical models and numerical electromagnetic simulations, we examine various geometric factors that can enhance the magnetic response, including the number of particles, spatial distribution, size, and shape. Our broad investigation provides researchers with analytical and numerical estimates of the magnetic response of nanoparticles, and the associated limits that can be expected. We found that a magnetic field enhancement comparable to the incident field requires very high magnetic susceptibilities, well beyond the values measured in functionalized gold nanoparticles thus far.