Nanoscale elemental and morphological imaging of nitrogen-fixing cyanobacteria.

Nitrogen-fixing cyanobacteria bind atmospheric nitrogen and carbon dioxide using sunlight. This experimental study focused on a laboratory-based model system, Anabaena sp., in nitrogen-depleted culture. When combined nitrogen is scarce, the filamentous procaryotes reconcile photosynthesis and nitrogen fixation by cellular differentiation into heterocysts. To better understand the influence of micronutrients on cellular function, 2D and 3D synchrotron X-ray fluorescence mappings were acquired from whole biological cells in their frozen-hydrated state at the Bionanoprobe, Advanced Photon Source. To study elemental homeostasis within these chain-like organisms, biologically relevant elements were mapped using X-ray fluorescence spectroscopy and energy-dispersive X-ray microanalysis. Higher levels of cytosolic K+, Ca2+, and Fe2+ were measured in the heterocyst than in adjacent vegetative cells, supporting the notion of elevated micronutrient demand. P-rich clusters, identified as polyphosphate bodies involved in nutrient storage, metal detoxification and osmotic regulation, were consistently co-localized with K+ and occasionally sequestered Mg2+, Ca2+, Fe2+, and Mn2+ ions. Machine-learning based k-mean clustering revealed that P/K clusters were associated with either Fe or Ca, with Fe and Ca clusters also occurring individually. In accordance with XRF nanotomography, distinct P/K-containing clusters close to the cellular envelope were surrounded by larger Ca-rich clusters. The transition metal Fe, which is part of nitrogenase enzyme, was detected as irregular shaped clusters. The elemental composition and cellular morphology of diazotrophic Anabaena sp. was visualized by multimodal imaging using AFM, SEM, and fluorescence microscopy. This paper discusses the first experimental results obtained with a combined in-line optical and X-ray fluorescence microscope at the Bionanoprobe.

Revealing chemistry-structure-function relationships in shark vertebrae across length scales.

Shark cartilage presents a complex material composed of collagen, proteoglycans, and bioapatite. In the present study, we explored the link between microstructure, chemical composition, and biomechanical function of shark vertebral cartilage using Polarized Light Microscopy (PLM), Atomic Force Microscopy (AFM), Confocal Raman Microspectroscopy, and Nanoindentation. Our investigation focused on vertebrae from Blacktip and Shortfin Mako sharks. As typical representatives of the orders Carcharhiniformes and Lamniformes, these species differ in preferred habitat, ecological role, and swimming style. We observed structural variations in mineral organization and collagen fiber arrangement using PLM and AFM. In both sharks, the highly calcified corpus calcarea shows a ridged morphology, while a chain-like network is present in the less mineralized intermedialia. Raman spectromicroscopy demonstrates a relative increase of glucosaminocycans (GAGs) with respect to collagen and a decrease in mineral-rich zones, underlining the role of GAGs in modulating bioapatite mineralization. Region-specific testing confirmed that intravertebral variations in mineral content and arrangement result in distinct nanomechanical properties. Local Young's moduli from mineralized regions exceeded bulk values by a factor of 10. Overall, this work provides profound insights into a flexible yet strong biocomposite, which is crucial for the extraordinary speed of cartilaginous fish in the worlds' oceans. STATEMENT OF SIGNIFICANCE: Shark cartilage is a morphologically complex material composed of collagen, sulfated proteoglycans, and calcium phosphate minerals. This study explores the link between microstructure, chemical composition, and biological mechanical function of shark vertebral cartilage at the micro- and nanometer scale in typical Carcharhiniform and Lamniform shark species, which represent different vertebral mineralization morphologies, swimming styles and speeds. By studying the intricacies of shark vertebrae, we hope to lay the foundation for biomimetic composite materials that harness lamellar reinforcement and tailored stiffness gradients, capable of dynamic and localized adjustments during movement.

Nanocrystalline iron hydroxide lignocellulose filters for arsenate remediation.

Harmful levels of environmental contaminants, such as arsenic (As), persist readily in the environment, threatening safe drinking water supplies in many parts of the world. In this paper, we present a straightforward and cost-effective filtration technology for the removal of arsenate from potable water. Biocomposite filters comprised of nanocrystalline iron oxides or oxyhydroxides mineralized within lignocellulose scaffolds constitute a promising low cost, low-tech avenue for the removal of these contaminants. Two types of iron oxide mineral phases, 2-line ferrihydrite (Fh) and magnetite (Mt), were synthesized within highly porous balsa wood using an environmentally benign modification process and studied in view of their effective removal of As from contaminated water. The mineral deposition pattern, minerology, as well as crystallinity, were assessed using scanning electron microscopy, transmission electron microscopy, micro-computed X-ray tomography, confocal Raman microscopy, infrared spectroscopy, and X-ray powder diffraction. Our results indicate a preferential distribution of the Fh mineral phase within the micro-porous cell wall and radial parenchyma cells of rays, while Mt is formed primarily at the cell wall/lumen interface of vessels and fibers. Water samples of known As concentrations were subjected to composite filters in batch incubation and gravity-driven flow-through adsorption tests. Eluents were analyzed using microwave plasma optical emission spectroscopy (MP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). By subjecting the filters to a flow of contaminated water, the time for As uptake was reduced to minutes rather than hours, while immobilizing the same amount of As. The retention of As within the composite filter was further confirmed through energy-dispersive X-ray mappings. Apart from addressing dangerously high levels of arsenate in potable water, these versatile iron oxide lignocellulosic filters harbor tremendous potential for addressing current and emerging environmental contaminants that are known to adsorb on iron oxide mineral phases, such as phosphate, polycyclic aromatic hydrocarbons or heavy metals.

Development of a Microfluidic Device for Exosome Isolation in Point-of-Care Settings.

Exosomes have gained recognition in cancer diagnostics and therapeutics. However, most exosome isolation methods are time-consuming, costly, and require bulky equipment, rendering them unsuitable for point-of-care (POC) settings. Microfluidics can be the key to solving these challenges. Here, we present a double filtration microfluidic device that can rapidly isolate exosomes via size-exclusion principles in POC settings. The device can efficiently isolate exosomes from 50-100 µL of plasma within 50 min. The device was compared against an already established exosome isolation method, polyethylene glycol (PEG)-based precipitation. The findings showed that both methods yield comparable exosome sizes and purity; however, exosomes isolated from the device exhibited an earlier miRNA detection compared to exosomes obtained from the PEG-based isolation. A comparative analysis of exosomes collected from membrane filters with 15 nm and 30 nm pore sizes showed a similarity in exosome size and miRNA detection, with significantly increased sample purity. Finally, TEM images were taken to analyze how the developed devices and PEG-based isolation alter exosome morphology and to analyze exosome sizes. This developed microfluidic device is cost-efficient and time-efficient. Thus, it is ideal for use in low-resourced and POC settings to aid in cancer and disease diagnostics and therapeutics.

Synchrotron-based X-ray fluorescence microscopy mapping the ionome of a toxic freshwater cyanobacterium.

Harmful algal blooms (HABs) pose a major environmental concern across the globe. In abundance, cyanobacteria, or so-called green-blue algae can produce extremely dangerous cyanotoxins that harm humans and animals. This study focused on the mapping and distribution of intracellular macro-and micronutrients of the wide-spread freshwater cyanobacteria Microcystis aeruginosa (M. aeruginosa). Towards a better understanding of trace metal uptake and homeostasis throughout the cell cycle, we quantitatively mapped the spatial distribution of the elements P, K, Fe, Ca, Zn, Mn, and Cu across the ultrastructure of frozen-hydrated single cells using state-of-the-art X-ray nanofluorescence imaging at the Advanced Photon Source (APS) at Argonne National Laboratory. Bulk cellular nutrient and trace metal content correlated well with the total intracellular elemental content in individual cells obtained by quantitative synchrotron X-ray fluorescence measurements. Multi-dimensional mappings showed P and K atoms colocalized as discrete semicircular hotspots that were analyzed with respect to their stoichiometry. Elevated Cu and Ca concentrations were detected along division plane of cells. P and K were found to have similar spatial elemental distribution with about 65% and 69% of the total cellular P and K, respectively, located at the hotspots. The P and K colocalization were refined further using nanotomography, showing a K envelope surrounding the P core. Inorganic P and organic P compounds were specified using solution-state 31P nuclear magnetic resonance (NMR) spectroscopy from M. aeruginosa. Of the total extracted P determined by 31P NMR spectroscopy, 47% were found to be nucleotides while only 11% were polyphosphates. Multimodal X-ray imaging provides a better understanding of intracellular biochemical processes in cyanobacteria, helping us monitor and combat an emerging environmental threat.

On stars and spikes: Resolving the skeletal morphology of planktonic Acantharia using synchrotron X-ray nanotomography and deep learning image segmentation.

Acantharia (Acantharea) are wide-spread marine protozoa, presenting one of the rare examples of strontium sulfate mineralization in the biosphere. Their endoskeletons consist of 20 spicules arranged according to a unique geometric pattern named Müller's principle. Given the diverse mineral architecture of the Acantharia class, we set out to examine the complex three-dimensional skeletal morphology at the nanometer scale using synchrotron X-ray nanotomography, followed by image segmentation based on deep learning methods. The present study focuses on how the spicules emanate from the robust central junction in the orders Symphyacanthida and Arthracanthida, the geometry of lateral spicule wings as well as pockets of interspicular space, which may be involved in cell compartmentalization. Through these morphometric studies, we observed subtle deviations from the previously described spatial arrangement of the spicules. According to our data, spicule shapes are adjusted in opposite spicules as to accommodate the overall spicule arrangement. In all types examined, previously unknown interspicular interstices were found in areas where radial spicules meet, which could have implications for the crystal growth mechanism and overall endoskeletal integrity. A deeper understanding of the spiculogenesis in Acantharia can provide biomimetic routes towards complex inorganic shapes. STATEMENT OF SIGNIFICANCE: Morphogenesis, the origin and control of shape, provides an avenue towards tailored inorganic materials. In this work, we explored the intricate skeletal organization of planktonic Acantharia, which are amongst the few strontium sulfate biomineralizing organisms in nature. By using nanoscale X-ray imaging and deep learning image segmentation, we found deviations from previously described geometric patterns and undiscovered skeletal features. The bio-inspired synthesis of inorganic materials with complex shape has important ramifications for solid-state chemistry and nanotechnology.

Effects of zinc and carnosine on aggregation kinetics of Amyloid-ß40 peptide.

The accumulation and amyloid formation of amyloid-ß (Aß) peptides is closely associated with the pathology of Alzheimer's disease. The physiological environment wherein Aß aggregation happens is crowded with a large variety of metal ions including Zn2+. In this study, we investigated the role of Zn2+ in regulating the aggregation kinetics of Aß40 peptide. Our results show that Zn2+ can shift a typical single sigmoidal aggregation kinetics of Aß40 to a biphasic aggregation process. Zn2+ aids in initiating the rapid self-assembly of monomers to form oligomeric intermediates, which further grow into amyloid fibrils in the first aggregation phase. The presence of Zn2+ also retards the appearance of the second aggregation phase in a concentration dependent manner. In addition, our results show that a natural dipeptide, carnosine, can greatly alleviate the effect of Zn2+ on Aß aggregation kinetics, most likely by coordinating with the metal ion to form chelates. These results suggest a potential in vivo protective effect of carnosine against the cytotoxicity of Aß by suppressing Zn2+-induced rapid formation of Aß oligomers.

Functional lignocellulosic materials prepared by ATRP from a wood scaffold.

Wood, a natural and abundant source of organic polymers, has been used as a scaffold to develop novel wood-polymer hybrid materials. Through a two-step surface-initiated Atom Transfer Radical Polymerization (ATRP), the porous wood structure can be effectively modified with polymer chains of various nature. In the present study, polystyrene and poly(N-isopropylacrylamide) were used. As shown with various characterization techniques including confocal Raman microscopy, FTIR, and SEM/EDX, the native wood ultrastructure and features are retained and the polymer chains can be introduced deep within the wood, i.e. inside the wood cell walls. The physical properties of the new materials have been studied, and results indicate that the insertion of polymer chains inside the wood cell wall alters the intrinsic properties of wood to yield a hybrid composite material with new functionalities. This approach to the functionalization of wood could lead to the fabrication of a new class of interesting functional materials and promote innovative utilizations of the renewable resource wood.

Hybrid wood materials with magnetic anisotropy dictated by the hierarchical cell structure.

Anisotropic and hierarchical structures are bound in nature and highly desired in engineered materials, due to their outstanding functions and performance. Mimicking such natural features with synthetic materials and methods has been a highly active area of research in the last decades. Unlike these methods, we use the native biomaterial wood, with its intrinsic anisotropy and hierarchy as a directional scaffold for the incorporation of magnetic nanoparticles inside the wood material. Nanocrystalline iron oxide particles were synthesized in situ via coprecipitation of ferric and ferrous ions within the interconnected pore network of bulk wood. Imaging with low-vacuum and cryogenic electron microscopy as well as spectral Raman mapping revealed layered nanosize particles firmly attached to the inner surface of the wood cell walls. The mineralogy of iron oxide was identified by XRD powder diffraction and Raman spectroscopy as a mixture of the spinel phases magnetite and maghemite. The intrinsic structural architecture of native wood entails a three-dimensional assembly of the colloidal iron oxide which results in direction-dependent magnetic features of the wood-mineral hybrid material. This superinduced magnetic anisotropy, as quantified by direction-dependent magnetic hysteresis loops and low-field susceptibility tensors, allows for directional lift, drag, alignment, (re)orientation, and actuation, and opens up novel applications of the natural resource wood.

Interaction of colloidal nanoparticles with their local environment: the (ionic) nanoenvironment around nanoparticles is different from bulk and determines the physico-chemical properties of the nanoparticles.

The physico-chemical properties of colloidal nanoparticles (NPs) are influenced by their local environment, as, in turn, the local environment influences the physico-chemical properties of the NPs. In other words, the local environment around NPs has a profound impact on the NPs, and it is different from bulk due to interaction with the NP surface. So far, this important effect has not been addressed in a comprehensive way in the literature. The vicinity of NPs can be sensitively influenced by local ions and ligands, with effects already occurring at extremely low concentrations. NPs in the Hückel regime are more sensitive to fluctuations in the ionic environment, because of a larger Debye length. The local ion concentration hereby affects the colloidal stability of the NPs, as it is different from bulk owing to Debye Hückel screening caused by the charge of the NPs. This can have subtle effects, now caused by the environment to the performance of the NP, such as for example a buffering effect caused by surface reaction on ultrapure ligand-free nanogold, a size quenching effect in the presence of specific ions and a significant impact on fluorophore-labelled NPs acting as ion sensors. Thus, the aim of this review is to clarify and give an unifying view of the complex interplay between the NP's surface with their nanoenvironment.

In situ non-DLVO stabilization of surfactant-free, plasmonic gold nanoparticles: effect of Hofmeister's anions.

Specific ion effects ranking in the Hofmeister sequence are ubiquitous in biochemical, industrial, and atmospheric processes. In this experimental study specific ion effects inexplicable by the classical DLVO theory have been investigated at curved water-metal interfaces of gold nanoparticles synthesized by a laser ablation process in liquid in the absence of any organic stabilizers. Notably, ion-specific differences in colloidal stability occurred in the Hückel regime at extraordinarily low salinities below 50 µM, and indications of a direct influence of ion-specific effects on the nanoparticle formation process are found. UV-vis, zeta potential, and XPS measurements help to elucidate coagulation properties, electrokinetic potential, and the oxidation state of pristine gold nanoparticles. The results clearly demonstrate that stabilization of ligand-free gold nanoparticles scales proportionally with polarizability and antiproportionally with hydration of anions located at defined positions in a direct Hofmeister sequence of anions. These specific ion effects might be due to the adsorption of chaotropic anions (Br(-), SCN(-), or I(-)) at the gold/water interface, leading to repulsive interactions between the partially oxidized gold particles during the nanoparticle formation process. On the other hand, kosmotropic anions (F(-) or SO4(2-)) seem to destabilize the gold colloid, whereas Cl(-) and NO3(-) give rise to an intermediate stability. Quantification of surface charge density indicated that particle stabilization is dominated by ion adsorption and not by surface oxidation. Fundamental insights into specific ion effects on ligand-free aqueous gold nanoparticles beyond purely electrostatic interactions are of paramount importance in biomedical or catalytic applications, since colloidal stability appears to depend greatly on the type of salt rather than on the amount.

Renewable and functional wood materials by grafting polymerization within cell walls.

A "grafting-from" polymerization approach within and at the complex and heterogeneous macromolecular assembly of wood cell walls is shown. The approach allows for the implementation of novel functionalities in renewable and functional wood-based materials. The native wood structure is retained and used as a hierarchical multiscale framework for a modular two-step polymerization process. The versatility and potential of the approach is shown by a polymerization of either hydrophobic or hydrophilic and pH-responsive monomers in the wood structure. Characterization of the modified wood reveals the presence of polymer in the cell wall, and the new properties of these wood materials are discussed.

Size control of laser-fabricated surfactant-free gold nanoparticles with highly diluted electrolytes and their subsequent bioconjugation.

Size control of laser-fabricated surfactant-free gold nanoparticles is a challenging endeavor. In this work, we show that size control can be achieved by adding ions with low salinity during synthesis. In addition, this approach offers the opportunity to fundamentally study ion interactions with bare nanoparticle surfaces and can help to elucidate the nanoparticle formation mechanism. The studies were carried out in a flow-through reactor and in the presence of NaCl, NaBr and sodium phosphate buffer at minimal ionic strengths. A significant size quenching effect at ionic strengths from 1-50 µM was found, which allowed surfactant-free nanoparticle size control with average diameters of 6-30 nm. This effect went along with low polydispersity and minimal aggregation tendencies and was confirmed by UV-vis spectroscopy, TEM, SEM and analytical disk centrifugation. Our findings indicate that size quenching originates from an anionic electrostatic stabilization depending on the nanoparticle surface area, which may be caused by specific ion adsorption. By subsequent delayed bioconjugation in liquid-flow using bovine serum albumin as a stabilizing agent, nano-bioconjugates with good stability in cell culture media were obtained, which are applicable in toxicology and cell biology.