Correlated cryo-SEM and CryoNanoSIMS imaging of biological tissue.

BACKGROUND: The development of nanoscale secondary ion mass spectrometry (NanoSIMS) has revolutionized the study of biological tissues by enabling, e.g., the visualization and quantification of metabolic processes at subcellular length scales. However, the associated sample preparation methods all result in some degree of tissue morphology distortion and loss of soluble compounds. To overcome these limitations an entirely cryogenic sample preparation and imaging workflow is required. RESULTS: Here, we report the development of a CryoNanoSIMS instrument that can perform isotope imaging of both positive and negative secondary ions from flat block-face surfaces of vitrified biological tissues with a mass- and image resolution comparable to that of a conventional NanoSIMS. This capability is illustrated with nitrogen isotope as well as trace element mapping of freshwater hydrozoan Green Hydra tissue following uptake of 15N-enriched ammonium. CONCLUSION: With a cryo-workflow that includes vitrification by high pressure freezing, cryo-planing of the sample surface, and cryo-SEM imaging, the CryoNanoSIMS enables correlative ultrastructure and isotopic or elemental imaging of biological tissues in their most pristine post-mortem state. This opens new horizons in the study of fundamental processes at the tissue- and (sub)cellular level. TEASER: CryoNanoSIMS: subcellular mapping of chemical and isotopic compositions of biological tissues in their most pristine post-mortem state.

A Resonant Graphene NEMS Vibrometer.

Measuring vibrations is essential to ensuring building structural safety and machine stability. Predictive maintenance is a central internet of things (IoT) application within the new industrial revolution, where sustainability and performance increase over time are going to be paramount. To reduce the footprint and cost of vibration sensors while improving their performance, new sensor concepts are needed. Here, double-layer graphene membranes are utilized with a suspended silicon proof demonstrating their operation as resonant vibration sensors that show outstanding performance for a given footprint and proof mass. The unveiled sensing effect is based on resonant transduction and has important implications for experimental studies involving thin nano and micro mechanical resonators that are excited by an external shaker.

Symbiotic nutrient exchange enhances the long-term survival of cassiosomes, the autonomous stinging-cell structures of Cassiopea.

Medusae of the widely distributed upside-down jellyfish Cassiopea release autonomous, mobile stinging structures. These so-called cassiosomes play a role in predator defense and prey capture, and are major contributors to "contactless" stinging incidents in (sub-)tropical shallow waters. While the presence of endosymbiotic dinoflagellates in cassiosomes has previously been observed, their potential contribution to the metabolism and long-term survival of cassiosomes is unknown. Combining stable isotope labeling and correlative scanning electron microscopy and nanoscale secondary ion mass spectrometry imaging with a long-term in vitro experiment, our study reveals a mutualistic symbiosis based on nutritional exchanges in dinoflagellate-bearing cassiosomes. We show that organic carbon input from the dinoflagellates fuels the metabolism of the host tissue and enables anabolic nitrogen assimilation. This symbiotic nutrient exchange enhances the life span of cassiosomes for at least one month in vitro. Overall, our study demonstrates that cassiosomes, in analogy with Cassiopea medusae, are photosymbiotic holobionts. Cassiosomes, which are easily accessible under aquarium conditions, promise to be a powerful new miniaturized model system for in-depth ultrastructural and molecular investigation of cnidarian photosymbioses.IMPORTANCEThe upside-down jellyfish Cassiopea releases autonomous tissue structures, which are a major cause of contactless stinging incidents in (sub-) tropical coastal waters. These so-called cassiosomes frequently harbor algal symbionts, yet their role in cassiosome functioning and survival is unknown. Our results show that cassiosomes are metabolically active and supported by algal symbionts. Algal photosynthesis enhances the cassiosomes long-term survival in the light. This functional understanding of cassiosomes thereby contributes to explaining the prevalence of contactless stinging incidents and the ecological success of some Cassiopea species. Finally, we show that cassiosomes are miniaturized symbiotic holobionts that can be used to study host-microbe interactions in a simplified system.

Electrostatic force microscopy as a broadly applicable method for characterizing pyroelectric materials.

A general method based on the combination of electrostatic force microscopy with thermal cycling of the substrate holder is presented for direct, nanoscale characterization of the pyroelectric effect in a range of materials and sample configurations using commercial atomic force microscope systems. To provide an example of its broad applicability, the technique was applied to the examination of natural tourmaline gemstones. The method was validated using thermal cycles similar to those experienced in ambient conditions, where the induced pyroelectric response produced localized electrostatic surface charges whose magnitude demonstrated a correlation with the iron content and heat dissipation of each gemstone variety. In addition, the surface charge was shown to persist even at thermal equilibrium. This behavior is attributed to constant, stochastic cooling of the gemstone surface through turbulent contact with the surrounding air and indicates a potential utility for energy harvesting in applications including environmental sensors and personal electronics. In contrast to previously reported methods, ours has a capacity to carry out such precise nanoscale measurements with little or no restriction on the sample of interest, and represents a powerful new tool for the characterization of pyroelectric materials and devices.

Integrated elastomer-based device for measuring the mechanics of adherent cell monolayers.

Cells in the body collectively sustain mechanical deformations in almost all physiological functions. From the morphogenesis stage, cells' ability to sustain stress is essential for the body's well-being. Several pathologies have been associated with abnormal mechanical properties, thus suggesting the Young's modulus as a biomarker to diagnose diseases and determine their progression. Advancements in the field are quite slow because current techniques for measuring cell and tissue mechanics rely on complex and bulky measurement platforms that have low repeatability rates and limited measurement time-scales. We present the first miniaturized system that allows accurate quantification of the Young's modulus of adherent cell monolayers over a longer time (1-2 days). Our approach is based on tensile testing and optical read-out. Thanks to a thoughtful design and material choice, we are able to miniaturize tensile testing platforms into a 1 cm × 2 cm device. We provide highly repeatable Young's modulus measurements in the relevant range between 3 kPa and 300 kPa, over time and under physiological conditions, thus representing an interesting alternative to existing measurement platforms. Furthermore, the compatibility with standard biological equipment, continuous optical imaging and measurements on all types of adherent cells make this device highly versatile. Measurements on human sarcoma osteogenic (SaOS2) and Madin-Darby canine kidney cells (MDCK) are reported. The demonstrated capability to measure real-time changes in mechanical properties, such as after chemical treatment, opens the door for investigating the effects of drugs on cell mechanics.

An ultra-fast mechanically active cell culture substrate.

We present a mechanically active cell culture substrate that produces complex strain patterns and generates extremely high strain rates. The transparent miniaturized cell stretcher is compatible with live cell microscopy and provides a very compact and portable alternative to other systems. A cell monolayer is cultured on a dielectric elastomer actuator (DEA) made of a 30 µm thick silicone membrane sandwiched between stretchable electrodes. A potential difference of several kV's is applied across the electrodes to generate electrostatic forces and induce mechanical deformation of the silicone membrane. The DEA cell stretcher we present here applies up to 38% tensile and 12% compressive strain, while allowing real-time live cell imaging. It reaches the set strain in well under 1 ms and generates strain rates as high as 870 s-1, or 87%/ms. With the unique capability to stretch and compress cells, our ultra-fast device can reproduce the rich mechanical environment experienced by cells in normal physiological conditions, as well as in extreme conditions such as blunt force trauma. This new tool will help solving lingering questions in the field of mechanobiology, including the strain-rate dependence of axonal injury and the role of mechanics in actin stress fiber kinetics.

Graphene MEMS: AFM probe performance improvement.

We explore the feasibility of growing a continuous layer of graphene in prepatterned substrates, like an engineered silicon wafer, and we apply this as a mold for the fabrication of AFM probes. This fabrication method proves the fabrication of SU-8 devices coated with graphene in a full-wafer parallel technology and with high yield. It also demonstrates that graphene coating enhances the functionality of SU-8 probes, turning them conductive and more resistant to wear. Furthermore, it opens new experimental possibilities such as studying graphene-graphene interaction at the nanoscale with the precision of an AFM or the exploration of properties in nonplanar graphene layers.

Emergent criticality in complex turing B-type atomic switch networks.

Recent advances in the neuromorphic operation of atomic switches as individual synapse-like devices demonstrate the ability to process information with both short-term and long-term memorization in a single two terminal junction. Here it is shown that atomic switches can be self-assembled within a highly interconnected network of silver nanowires similar in structure to Turing's "B-Type unorganized machine", originally proposed as a randomly connected network of NAND logic gates. In these experimental embodiments,complex networks of coupled atomic switches exhibit emergent criticality similar in nature to previously reported electrical activity of biological brains and neuron assemblies. Rapid fluctuations in electrical conductance display metastability and power law scaling of temporal correlation lengths that are attributed to dynamic reorganization of the interconnected electro-ionic network resulting from induced non-equilibrium thermodynamic instabilities. These collective properties indicate a potential utility for realtime,multi-input processing of distributed sensory data through reservoir computation. We propose these highly coupled, nonlinear electronic networks as an implementable hardware-based platform toward the creation of physically intelligent machines.

Neuromorphic atomic switch networks.

Efforts to emulate the formidable information processing capabilities of the brain through neuromorphic engineering have been bolstered by recent progress in the fabrication of nonlinear, nanoscale circuit elements that exhibit synapse-like operational characteristics. However, conventional fabrication techniques are unable to efficiently generate structures with the highly complex interconnectivity found in biological neuronal networks. Here we demonstrate the physical realization of a self-assembled neuromorphic device which implements basic concepts of systems neuroscience through a hardware-based platform comprised of over a billion interconnected atomic-switch inorganic synapses embedded in a complex network of silver nanowires. Observations of network activation and passive harmonic generation demonstrate a collective response to input stimulus in agreement with recent theoretical predictions. Further, emergent behaviors unique to the complex network of atomic switches and akin to brain function are observed, namely spatially distributed memory, recurrent dynamics and the activation of feedforward subnetworks. These devices display the functional characteristics required for implementing unconventional, biologically and neurally inspired computational methodologies in a synthetic experimental system.

Oxide nanocrystal based nanocomposites for fabricating photoplastic AFM probes.

We report on the synthesis, characterization and application of a novel nanocomposite made of a negative tone epoxy based photoresist modified with organic-capped Fe(2)O(3) nanocrystals (NCs). The mechanical properties of the nanocomposite drastically improve upon incorporation of a suitable concentration of NCs in the polymer, without deteriorating its photolithography performance. High aspect ratio 3D microstructures made of the nanocomposite have been fabricated with a uniform surface morphology and with a resolution down to few micrometres. The embedded organic-capped Fe(2)O(3) NCs drastically increase the stiffness and hardness of the epoxy based photoresist matrix, making the final material extremely interesting for manufacturing miniaturized polymer based mechanical devices and systems. In particular, the nanocomposite has been used as structural material for fabricating photoplastic Atomic Force Microscopy (AFM) probes with integrated tips showing outstanding mechanical response and high resolution imaging performance. The fabricated probes consist of straight cantilevers with low stress-gradient and high quality factors, incorporating sharp polymeric tips. They present considerably improved performance compared to pure epoxy based photoresist AFM probes, and to commercial silicon AFM probes.