3D Printing Hierarchically Nano-Ordered Structures.

Natural materials are composed of a limited number of molecular building blocks and their exceptional properties are governed by their hierarchical structure. However, this level of precision is unattainable with current state-of-the-art materials for 3D printing. Herein, new self-assembled printable materials based on block copolymers (BCPs) enabling precise control of the nanostructure in 3D are presented. In particular, well-defined BCPs consisting of poly(styrene) (PS) and a polymethacrylate-based copolymer decorated with printable units are selected as suitable self-assembled materials and synthesized using controlled radical polymerization. The synthesized library of BCPs are utilized as printable formulations for the fabrication of complex 3D microstructures using two-photon laser printing. By fine-tuning the BCP composition and solvent in the formulations, the fabrication of precise 3D nano-ordered structures is demonstrated for the first time. A key point of this work is the achievement of controlled nano-order within the entire 3D structures. Thus, imaging of the cross-sections of the 3D printed samples is performed, enabling the visualization also from the inside. The presented versatile approach is expected to create new avenues for the precise design of functional polymer materials suitable for high-resolution 3D printing exhibiting tailor-made nanostructures.

Electron-beam lithography of cinnamate polythiophene films: conductive nanorods for electronic applications.

We report the electron-beam induced crosslinking of cinnamate-substituted polythiophene proceeding via excited state [2+2]-cycloaddition. Network formation in thin films is evidenced by infrared spectroscopy and film retention experiments. For the polymer studied herin, the electron-stimulated process appears to be superior to photo (UV)-induced crosslinking as it leads to less degradation. Electron beam lithography (EBL) patterns cinnamate-substituted polythiophene thin films on the nanoscale with a resolution of around 100 nm. As a proof of concept, we fabricated nanoscale organic transistors using doped and cross-linked P3ZT as contact fingers in thin film transistors.

Analysis of the optical properties of the silvery spots on the wings of the Gulf Fritillary, Dione vanillae.

The ventral face of the wings of the butterfly Dione vanillae is covered with bright and shiny silvery spots. These areas contain densely packed ground- and coverscales with a bright metallic appearance reflecting more than 50% of light uniformly over the visible range. Our analysis shows that this optically attractive feature is caused by the inner microstructure of the scales located in these areas. Electron microscopy of cross sections through the scales shows that upper and lower lamina, supporting trabeculae, and topping ridges can be approximated by a 'circus tent'-like geometry. By simulating its optical properties, we show that a moderate disorder of this geometry is important for the uniform reflection of light resulting in the silvery appearance.

Overlapping Properties of the Short Membrane-Active Peptide BP100 With (i) Polycationic TAT and (ii) α-helical Magainin Family Peptides.

BP100 is a short, designer-made membrane-active peptide with multiple functionalities: antimicrobial, cell-penetrating, and fusogenic. Consisting of five lysines and 6 hydrophobic residues, BP100 was shown to bind to lipid bilayers as an amphipathic α-helix, but its mechanism of action remains unclear. With these features, BP100 embodies the characteristics of two distinctly different classes of membrane-active peptides, which have been studied in detail and where the mechanism of action is better understood. On the one hand, its amphiphilic helical structure is similar to the pore forming magainin family of antimicrobial peptides, though BP100 is much too short to span the membrane. On the other hand, its length and high charge density are reminiscent of the HIV-TAT family of cell penetrating peptides, for which inverted micelles have been postulated as translocation intermediates, amongst other mechanisms. Assays were performed to test the antimicrobial and hemolytic activity, the induced leakage and fusion of lipid vesicles, and cell uptake. From these results the functional profiles of BP100, HIV-TAT, and the magainin-like peptides magainin 2, PGLa, MSI-103, and MAP were determined and compared. It is observed that the activity of BP100 resembles most closely the much longer amphipathic α-helical magainin-like peptides, with high antimicrobial activity along with considerable fusogenic and hemolytic effects. In contrast, HIV-TAT shows almost no antimicrobial, fusogenic, or hemolytic effects. We conclude that the amphipathic helix of BP100 has a similar membrane-based activity as magainin-like peptides and may have a similar mechanism of action.

Microengineered Hollow Graphene Tube Systems Generate Conductive Hydrogels with Extremely Low Filler Concentration.

The fabrication of electrically conductive hydrogels is challenging as the introduction of an electrically conductive filler often changes mechanical hydrogel matrix properties. Here, we present an approach for the preparation of hydrogel composites with outstanding electrical conductivity at extremely low filler loadings (0.34 S m-1, 0.16 vol %). Exfoliated graphene and polyacrylamide are microengineered to 3D composites such that conductive graphene pathways pervade the hydrogel matrix similar to an artificial nervous system. This makes it possible to combine both the exceptional conductivity of exfoliated graphene and the adaptable mechanical properties of polyacrylamide. The demonstrated approach is highly versatile regarding porosity, filler material, as well as hydrogel system. The important difference to other approaches is that we keep the original properties of the matrix, while ensuring conductivity through graphene-coated microchannels. This novel approach of generating conductive hydrogels is very promising, with particular applications in the fields of bioelectronics and biohybrid robotics.

Fast Response Organic Supramolecular Transistors Utilizing In-Situ π-Ion Gels.

Despite their remarkable charge carrier mobility when forming well-ordered fibers, supramolecular transistors often suffer from poor processability that hinders device integration, resulting in disappointing transconductance and output currents. Here, a new class of supramolecular transistors, π-ion gel transistors (PIGTs), is presented. An in situ π-ion gel, which is an unprecedented composite of semiconducting nanofibers and an enclosed ionic liquid, is directly employed as an active material and internal capacitor. In comparison to other supramolecular transistors, a PIGT displays a high transconductance (133 µS) and output current (139 µA at -6 V), while retaining a high charge-carrier mobility (4.2 × 10-2 cm2 V-1 s-1 ) and on/off ratio (3.7 × 104 ). Importantly, the unique device configuration and the high ionic conductivity associated with the distinct nanosegregation enables the fastest response among accumulation-mode electrochemical-based transistors (<20 µs). Considering the advantages of the absence of dielectric layers and the facile fabrication process, PIGT has great potential to be utilized in printed flexible devices. The device platform is widely applicable to various supramolecular assemblies, shedding light on the interdisciplinary research of supramolecular chemistry and organic electronics.

3D Two-Photon Microprinting of Nanoporous Architectures.

A photoresist system for 3D two-photon microprinting is presented, which enables the printing of inherently nanoporous structures with mean pore sizes around 50 nm by means of self-organization on the nanoscale. A phase separation between polymerizable and chemically inert photoresist components leads to the formation of 3D co-continuous structures. Subsequent washing-out of the unpolymerized phase reveals the porous polymer structures. To characterize the volume properties of the printed structures, scanning electron microscopy images are recorded from ultramicrotome sections. In addition, the light-scattering properties of the 3D-printed material are analyzed. By adjusting the printing parameters, the porosity can be controlled during 3D printing. As an application example, a functioning miniaturized Ulbricht light-collection sphere is 3D printed and tested.

Siloxane-functionalised surface patterns as templates for the ordered deposition of thin lamellar objects.

A novel method is demonstrated for ordered deposition of thin lamellar objects from a liquid environment onto solid substrates by solid/fluid/solid-driven organisation. Surface functionalisation forms a template pattern that accumulates the lamellar objects by site-selective wetting of the target area without the need for a physical fluid containment. Contrary to conventional handling methods, no mechanical contact occurs, which facilitates the ordered deposition without wrinkles or ruptures. An additive and a subtractive process for the creation of such templates are presented. The subtractive process starts with the complete silanisation of the substrate in the vapour phase followed by site-selective oxygen plasma treatment of the siloxane film. The additive process uses microcontact printing to transfer the target pattern. Both processes are characterised by optical inspection of the wetting contours and it is found that site-selective plasma treatment shows a better pattern fidelity. The patterns obtained by site-selective plasma treatment are also subject to ToF-SIMS analysis and show good chemical contrast between hydrophilic and hydrophobic areas. The ordered deposition of lamellar objects by this new method is demonstrated for 60 nm thick ultramicrotome sections of epoxide resin on pre-patterned glass substrates.

Printing Poly( p-phenyleneethynylene) PLEDs.

Gravure printing of functional thin-film layers of side-chain-substituted poly( para-phenyleneethynylene)s (PPEs) is reported. Rheological properties of PPEs in combination with the Hansen solubility model allowed the formulation of enhanced single-component inks. Layer evaluation is performed with reflectometric thin-film recordings in an optical setup for laterally resolved large-area investigation using imaging color reflectometry. An organic light-emitting diode in a simple glass/indium tin oxide/poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)/PPE/LiF-Al stack was gravure-printed from the improved ink showing excellent luminance (542 cd m-2, U = 11.5 V) for this polymer class.

Novel Functional TPE Polymers: Aggregation-Induced Emission, pH Response, and Solvatochromic Behavior.

Four tetraphenylethylene (TPE)-based aryleneethynylene polymers with amino or nitro groups are reported. They display strong aggregation-induced emission (AIE). The functional groups trigger acidochromic changes in the emission behavior of these polymers. Amino-substituted P1-P3 exhibit pH response through protonation of the amino groups. The position of the amino groups (on TPE or the side chains) influences the fluorescence intensity or emission wavelength as a response to different pH values. Nitro-P4 is solvatochromic due to its donor-acceptor structure. AIE, intramolecular charge transfer, and Förster resonance energy transfer define the fluorescence-based performance of the polymers. The amino-functionalized TPE polymers show excellent nitroarene-sensing performance. P4 is less effective than the amino polymers. A sensor array based on P1-P3 identifies 12 different nitroarenes in water.

Dipole-Switchable Poly(para-phenyleneethynylene)s: Ferroelectric Conjugated Polymers.

Dipolar 2,3-difluorobenzene units are introduced into the backbone of a poly(para-phenyleneethynylene) (PPE) to generate a ferroelectric conjugated polymer. The structural features of the partially fluorinated PPE allow for the generation of a remanent polarization in the solid state; the difluorinated benzene rings behave as molecular rotors at high temperature, while at room temperature, stacking of such rings clamps down the ring orientation. The molecular dipoles can still be oriented by moderate external electrical fields at room temperature, and this PPE is then ferroelectric. The concept should be transferable to other poly(aryleneethynylene)s, and novel conjugated ferroelectric conjugated polymers will be accessible using this concept.

Modern field emission scanning electron microscopy provides new perspectives for imaging kidney ultrastructure.

Recent progress in electron microscopy (EM) techniques has opened new pathways to study renal tissue in research and pathology. Modern field emission scanning EM may be utilized to scan thin sections of resin-embedded tissue mounted on a conductive support. Here we sought to achieve automated imaging without the typical limitations of transmission EM with equivalent or superior quality. Extended areas of tissue were either imaged in two (nanotomy) or in three dimensions (volume EM) by serial-section-based array tomography. Single-beam and fast-recording multi-beam field emission scanning EM instruments were compared using perfusion-fixed rodent kidneys. High-resolution scans produced excellent images of tissue, cells, and organelles down to macromolecular complexes. Digital stitching of image tiles in both modes allowed seamless Google Earth-like zooming from overview to regions of interest at the nanoscale. Large datasets were created that can be rapidly shared between scientists of different disciplines or pathologists using open source software. Three-dimensional array tomography of thin sections was followed by segmentation to visualize selected features in a large volume. Furthermore, correlative light-EM enabled the identification of functional information in a structural context. Thus, limitations in biomedical transmission EM can be overcome by introducing field emission scanning EM-based technology that permits high-quality, large field-of-view nanotomy, volume EM, and correlative light-EM modes. Advantages of virtual microscopy in clinical and experimental nephrology are illustrated.

New variant of necklace fibres display peculiar lysosomal structures and mitophagy.

Here, we describe a new variant of necklace fibres with specific myopathological features that have not been described thus far. They were observed in two patients, from two independent families with identical DNM2 (dynamin 2) mutation (c.1106 G > A (p.Arg369Gln)), displaying mildly heterogeneous clinical phenotypes. The variant is characterized by lysosomal inclusions, arranged in a necklace pattern, containing homogenous material, devoid of myonuclei. The so-called necklace region has a certain characteristic distance to the sarcolemma. Electron microscopy, including three dimensional reconstructions of serial section images highlights their ultrastructural properties and relation to neighbouring organelles. This new pattern is compared to the previously reported patterns in muscle biopsies containing necklace fibres associated with MTM1- and DNM2-mutations.

Discrete Triptycene-Based Hexakis(metalsalphens): Extrinsic Soluble Porous Molecules of Isostructural Constitution.

In 2013 the concept of OMIMs (organic molecules of intrinsic microporosity) was introduced by McKeown et al. These OMIMs are constructed on the basis of rigid molecular cores such as triptycene, spirobifluorenes, and others. Like shape-persistent organic cages, these are soluble discrete molecules and therefore an interesting alternative to 3D, insoluble porous materials, such as metal-organic frameworks, covalent-organic frameworks, or zeolites. OMIMs are chemically and thermally robust because the formation of strong covalent bonds has been used for their synthesis. To date, a few OMIMs have been reported, though most of them did not contain any functional unit to enhance gas sorption properties. This work introduces an isostructural series of metal-salphene based OMIMs with different metal ions (Zn2+ , Ni2+ , Cu2+ , Pd2+ , and Pt2+ ) integrated into the backbone. The influence of the metal centers on interaction with gas molecules has been investigated by gas sorption experiments.

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

Optical Properties and Sequence Information of Tin-Centered Conjugated Microporous Polymers.

We synthesized conjugated microporous polymers (CMPs) based on tetrakis(4-ethynylphenyl)stannane and diiodobenzene as tectons, using Sonogashira couplings under different conditions. Through variation of the reaction conditions (catalysts, bases and solvents), appearance, surface area and emission properties of the formed CMPs were significantly altered. Wet-chemical, acid-mediated digestion and analysis of the resulting struts of these otherwise insoluble networks give insight into the molecular setup.

Transforming a chemically labile [2+3] imine cage into a robust carbamate cage.

The synthesis of a highly acid and base-stable shape-persistent porous carbamate cage is presented. This cage is stable even in hot (100 °C) 1 M hydrochloric acid (pH = 0) or in concentrated hydrochloric acid (pH = -1) at room temperature without decomposition.

Cell Fixation by Light-Triggered Release of Glutaraldehyde.

Chemical fixation of living cells for microscopy is commonly achieved by crosslinking of intracellular proteins with dialdehydes prior to examination. We herein report a photocleavable protecting group for glutaraldehyde that results in a light-triggered and membrane-permeable fixative, which is nontoxic prior to photocleavage. Lipophilic ester groups allow for diffusion across the cell membrane and intracellular accumulation after enzymatic hydrolysis. Irradiation with UV light releases glutaraldehyde. The in situ generated fixative crosslinks intracellular proteins and preserves and stabilizes the cell so that it is ready for microscopy. In contrast to conventional glutaraldehyde fixation, tissue autofluorescence does not increase after fixation. Caged glutaraldehyde may in future enable functional experiments on living cells under a light microscope in which events of interest can be stopped in spatially confined volumes at defined time points. Samples with individually stopped events could then later be analyzed in ultrastructural studies.

Truxene-Based Hyperbranched Conjugated Polymers: Fluorescent Micelles Detect Explosives in Water.

We report two hyperbranched conjugated polymers (HCP) with truxene units as core and 1,4-didodecyl-2,5-diethynylbenzene as well as 1,4-bis(dodecyloxy)-2,5-diethynylbenzene as comonomers. Two analogous poly(para-phenyleneethynylene)s (PPE) are also prepared as comparison to demonstrate the difference between the truxene and the phenyl moieties in their optical properties and their sensing performance. The four polymers are tested for nitroaromatic analytes and display different fluorescence quenching responses. The quenching efficiencies are dependent upon the spectral overlap between the absorbance of the analyte and the emission of the fluorescent polymer. Optical fingerprints are obtained, based on the unique response patterns of the analytes toward the polymers. With this small sensor array, one can distinguish nine nitroaromatic analytes with 100% accuracy. The amphiphilic polymer F127 (a polyethylene glycol-polypropylene glycol block copolymer) carries the hydrophobic HCPs and self-assembles into micelles in water, forming highly fluorescent HCP micelles. The micelle-bound conjugated polymers detect nitroaromatic analytes effectively in water and show an increased sensitivity compared to the sensing of nitroaromatics in organic solvents. The nitroarenes are also discriminated in water using this four-element chemical tongue.

Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues.

BACKGROUND: Imaging large volumes such as entire cells or small model organisms at nanoscale resolution seemed an unrealistic, rather tedious task so far. Now, technical advances have lead to several electron microscopy (EM) large volume imaging techniques. One is array tomography, where ribbons of ultrathin serial sections are deposited on solid substrates like silicon wafers or glass coverslips. RESULTS: To ensure reliable retrieval of multiple ribbons from the boat of a diamond knife we introduce a substrate holder with 7 axes of translation or rotation specifically designed for that purpose. With this device we are able to deposit hundreds of sections in an ordered way in an area of 22 × 22 mm, the size of a coverslip. Imaging such arrays in a standard wide field fluorescence microscope produces reconstructions with 200 nm lateral resolution and 100 nm (the section thickness) resolution in z. By hierarchical imaging cascades in the scanning electron microscope (SEM), using a new software platform, we can address volumes from single cells to complete organs. In our first example, a cell population isolated from zebrafish spleen, we characterize different cell types according to their organelle inventory by segmenting 3D reconstructions of complete cells imaged with nanoscale resolution. In addition, by screening large numbers of cells at decreased resolution we can define the percentage at which different cell types are present in our preparation. With the second example, the root tip of cress, we illustrate how combining information from intermediate resolution data with high resolution data from selected regions of interest can drastically reduce the amount of data that has to be recorded. By imaging only the interesting parts of a sample considerably less data need to be stored, handled and eventually analysed. CONCLUSIONS: Our custom-designed substrate holder allows reproducible generation of section libraries, which can then be imaged in a hierarchical way. We demonstrate, that EM volume data at different levels of resolution can yield comprehensive information, including statistics, morphology and organization of cells and tissue. We predict, that hierarchical imaging will be a first step in tackling the big data issue inevitably connected with volume EM.