High Aspect Ratio Nanoscale Pores through BCP-Based Metal Oxide Masks and Advanced Dry Etching.

The reliable and regular modification of the surface properties of substrates plays a crucial role in material research and the development of functional surfaces. A key aspect of this is the development of the surface pores and topographies. These can confer specific advantages such as high surface area as well as specific functions such as hydrophobic properties. Here, we introduce a combination of nanoscale self-assembled block-copolymer-based metal oxide masks with optimized deep reactive ion etching (DRIE) of silicon to permit the fabrication of porous topographies with aspect ratios of up to 50. Following the evaluation of our procedure and involved parameters using various techniques, such as AFM or SEM, the suitability of our features for applications relying on high light absorption as well as efficient thermal management is explored and discussed in further detail.

ERnet: a tool for the semantic segmentation and quantitative analysis of endoplasmic reticulum topology.

The ability to quantify structural changes of the endoplasmic reticulum (ER) is crucial for understanding the structure and function of this organelle. However, the rapid movement and complex topology of ER networks make this challenging. Here, we construct a state-of-the-art semantic segmentation method that we call ERnet for the automatic classification of sheet and tubular ER domains inside individual cells. Data are skeletonized and represented by connectivity graphs, enabling precise and efficient quantification of network connectivity. ERnet generates metrics on topology and integrity of ER structures and quantifies structural change in response to genetic or metabolic manipulation. We validate ERnet using data obtained by various ER-imaging methods from different cell types as well as ground truth images of synthetic ER structures. ERnet can be deployed in an automatic high-throughput and unbiased fashion and identifies subtle changes in ER phenotypes that may inform on disease progression and response to therapy.

Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices.

The culturing and investigation of individual marine specimens in lab environments is crucial to further our understanding of this highly complex ecosystem. However, the obtained results and their relevance are often limited by a lack of suitable experimental setups enabling controlled specimen growth in a natural environment while allowing for precise monitoring and in-depth observations. In this work, we explore the viability of a microfluidic device for the investigation of the growth of the alga Saccharina latissima to enable high-resolution imaging by confining the samples, which usually grow in 3D, to a single 2D plane. We evaluate the specimen's health based on various factors such as its growth rate, cell shape, and major developmental steps with regard to the device's operating parameters and flow conditions before demonstrating its compatibility with state-of-the-art microscopy imaging technologies such as the skeletonisation of the specimen through calcofluor white-based vital staining of its cell contours as well as the immunolocalisation of the specimen's cell wall. Furthermore, by making use of the on-chip characterisation capabilities, we investigate the influence of altered environmental illuminations on the embryonic development using blue and red light. Finally, live tracking of fluorescent microspheres deposited on the surface of the embryo permits the quantitative characterisation of growth at various locations of the organism.

Mechanical factors contributing to the Venus flytrap's rate-dependent response to stimuli.

The sensory hairs of the Venus flytrap (Dionaea muscipula Ellis) detect mechanical stimuli imparted by their prey and fire bursts of electrical signals called action potentials (APs). APs are elicited when the hairs are sufficiently stimulated and two consecutive APs can trigger closure of the trap. Earlier experiments have identified thresholds for the relevant stimulus parameters, namely the angular displacement [Formula: see text] and angular velocity [Formula: see text]. However, these experiments could not trace the deformation of the trigger hair's sensory cells, which are known to transduce the mechanical stimulus. To understand the kinematics at the cellular level, we investigate the role of two relevant mechanical phenomena: viscoelasticity and intercellular fluid transport using a multi-scale numerical model of the sensory hair. We hypothesize that the combined influence of these two phenomena and [Formula: see text] contribute to the flytrap's rate-dependent response to stimuli. In this study, we firstly perform sustained deflection tests on the hair to estimate the viscoelastic material properties of the tissue. Thereafter, through simulations of hair deflection tests at different loading rates, we were able to establish a multi-scale kinematic link between [Formula: see text] and the cell wall stretch [Formula: see text]. Furthermore, we find that the rate at which [Formula: see text] evolves during a stimulus is also proportional to [Formula: see text]. This suggests that mechanosensitive ion channels, expected to be stretch-activated and localized in the plasma membrane of the sensory cells, could be additionally sensitive to the rate at which stretch is applied.

Embedded Microbubbles for Acoustic Manipulation of Single Cells and Microfluidic Applications.

Acoustically excited microstructures have demonstrated significant potential for small-scale biomedical applications by overcoming major microfluidic limitations. Recently, the application of oscillating microbubbles has demonstrated their superiority over acoustically excited solid structures due to their enhanced acoustic streaming at low input power. However, their limited temporal stability hinders their direct applicability for industrial or clinical purposes. Here, we introduce the embedded microbubble, a novel acoustofluidic design based on the combination of solid structures (poly(dimethylsiloxane)) and microbubbles (air-filled cavity) to combine the benefits of both approaches while minimizing their drawbacks. We investigate the influence of various design parameters and geometrical features through numerical simulations and experimentally evaluate their manipulation capabilities. Finally, we demonstrate the capabilities of our design for microfluidic applications by investigating its mixing performance as well as through the controlled rotational manipulation of individual HeLa cells.

Reduced Etch Lag and High Aspect Ratios by Deep Reactive Ion Etching (DRIE).

Deep reactive ion etching (DRIE) with the Bosch process is one of the key procedures used to manufacture micron-sized structures for MEMS and microfluidic applications in silicon and, hence, of increasing importance for miniaturisation in biomedical research. While guaranteeing high aspect ratio structures and providing high design flexibility, the etching procedure suffers from reactive ion etching lag and often relies on complex oxide masks to enable deep etching. The reactive ion etching lag, leading to reduced etch depths for features exceeding an aspect ratio of 1:1, typically causes a height difference of above 10% for structures with aspect ratios ranging from 2.5:1 to 10:1, and, therefore, can significantly influence subsequent device functionality. In this work, we introduce an optimised two-step Bosch process that reduces the etch lag to below 1.5%. Furthermore, we demonstrate an improved three-step Bosch process, allowing the fabrication of structures with 6 µm width at depths up to 180 µm while maintaining their stability.

3D mechanical characterization of single cells and small organisms using acoustic manipulation and force microscopy.

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems.

A single touch can provide sufficient mechanical stimulation to trigger Venus flytrap closure.

The carnivorous Venus flytrap catches prey by an ingenious snapping mechanism. Based on work over nearly 200 years, it has become generally accepted that two touches of the trap's sensory hairs within 30 s, each one generating an action potential, are required to trigger closure of the trap. We developed an electromechanical model, which, however, suggests that under certain circumstances one touch is sufficient to generate two action potentials. Using a force-sensing microrobotic system, we precisely quantified the sensory-hair deflection parameters necessary to trigger trap closure and correlated them with the elicited action potentials in vivo. Our results confirm the model's predictions, suggesting that the Venus flytrap may be adapted to a wider range of prey movements than previously assumed.

Kinematics Governing Mechanotransduction in the Sensory Hair of the Venus flytrap.

Insects fall prey to the Venus flytrap (Dionaea muscipula) when they touch the sensory hairs located on the flytrap lobes, causing sudden trap closure. The mechanical stimulus imparted by the touch produces an electrical response in the sensory cells of the trigger hair. These cells are found in a constriction near the hair base, where a notch appears around the hair's periphery. There are mechanosensitive ion channels (MSCs) in the sensory cells that open due to a change in membrane tension; however, the kinematics behind this process is unclear. In this study, we investigate how the stimulus acts on the sensory cells by building a multi-scale hair model, using morphometric data obtained from µ-CT scans. We simulated a single-touch stimulus and evaluated the resulting cell wall stretch. Interestingly, the model showed that high stretch values are diverted away from the notch periphery and, instead, localized in the interior regions of the cell wall. We repeated our simulations for different cell shape variants to elucidate how the morphology influences the location of these high-stretch regions. Our results suggest that there is likely a higher mechanotransduction activity in these 'hotspots', which may provide new insights into the arrangement and functioning of MSCs in the flytrap.

Feeling the force: how pollen tubes deal with obstacles.

Physical forces are involved in the regulation of plant development and morphogenesis by translating mechanical stress into the modification of physiological processes, which, in turn, can affect cellular growth. Pollen tubes respond rapidly to external stimuli and provide an ideal system to study the effect of mechanical cues at the single-cell level. Here, pollen tubes were exposed to mechanical stress while monitoring the reconfiguration of their growth and recording the generated forces in real-time. We combined a lab-on-a-chip device with a microelectromechanical systems (MEMS)-based capacitive force sensor to mimic and quantify the forces that are involved in pollen tube navigation upon confronting mechanical obstacles. Several stages of obstacle avoidance were identified, including force perception, growth adjustment and penetration. We have experimentally determined the perceptive force threshold, which is the force threshold at which the pollen tube reacts to an obstacle, for Lilium longiflorum and Arabidopsis thaliana. In addition, the method we developed provides a way to calculate turgor pressure based on force and optical data. Pollen tubes sense physical barriers and actively adjust their growth behavior to overcome them. Furthermore, our system offers an ideal platform to investigate intracellular activity during force perception and growth adaption in tip growing cells.

Neutrophil-inspired propulsion in a combined acoustic and magnetic field.

Systems capable of precise motion in the vasculature can offer exciting possibilities for applications in targeted therapeutics and non-invasive surgery. So far, the majority of the work analysed propulsion in a two-dimensional setting with limited controllability near boundaries. Here we show bio-inspired rolling motion by introducing superparamagnetic particles in magnetic and acoustic fields, inspired by a neutrophil rolling on a wall. The particles self-assemble due to dipole-dipole interaction in the presence of a rotating magnetic field. The aggregate migrates towards the wall of the channel due to the radiation force of an acoustic field. By combining both fields, we achieved a rolling-type motion along the boundaries. The use of both acoustic and magnetic fields has matured in clinical settings. The combination of both fields is capable of overcoming the limitations encountered by single actuation techniques. We believe our method will have far-reaching implications in targeted therapeutics.Devising effective swimming and propulsion strategies in microenvironments is attractive for drug delivery applications. Here Ahmed et al. demonstrate a micropropulsion strategy in which a combination of magnetic and acoustic fields is used to assemble and propel colloidal particles along channel walls.

Correction: High precision, localized proton gradients and fluxes generated by a microelectrode device induce differential growth behaviors of pollen tubes.

Correction for 'High precision, localized proton gradients and fluxes generated by a microelectrode device induce differential growth behaviors of pollen tubes' by Chengzhi Hu et al., Lab Chip, 2017, 17, 671-680.

High precision, localized proton gradients and fluxes generated by a microelectrode device induce differential growth behaviors of pollen tubes.

Pollen tubes are tip-growing plant cells that deliver the sperm cells to the ovules for double fertilization of the egg cell and the endosperm. Various directional cues can trigger the reorientation of pollen tube growth direction on their passage through the female tissues. Among the external stimuli, protons serve an important, regulatory role in the control of pollen tube growth. The generation of local guidance cues has been challenging when investigating the mechanisms of perception and processing of such directional triggers in pollen tubes. Here, we developed and characterized a microelectrode device to generate a local proton gradient and proton flux through water electrolysis. We confirmed that the cytoplasmic pH of pollen tubes varied with environmental pH change. Depending on the position of the pollen tube tip relative to the proton gradient, we observed alterations in the growth behavior, such as bursting at the tip, change in growth direction, or complete growth arrest. Bursting and growth arrest support the hypothesis that changes in the extracellular H+ concentration may interfere with cell wall integrity and actin polymerization at the growing tip. A change in growth direction for some pollen tubes implies that they can perceive the local proton gradient and respond to it. We also showed that the growth rate is directly correlated with the extracellular pH in the tip region. Our microelectrode approach provides a simple method to generate protons and investigate their effect on plant cell growth.

Probing the micromechanics of the fastest growing plant cell - the pollen tube.

The pollen tube is a fast growing cellular protrusion that plays a key role in the reproductive process of flowering plants. It serves as an important model for studying cellular morphogenesis, anisotropic growth mechanisms, and cellular signaling in the plant sciences. The anisotropic growth of pollen tubes is driven by a finely tuned control of the intracellular turgor pressure and the extensibility of the cell wall. To decipher this internal feedback loop and mathematically model the growth process, a quantitative understanding of the mechanical properties of the cell wall is crucial, in addition to biochemical investigations. We report an integrated microfluidic-MEMS force sensor system that allows for high-throughput optical and mechanical investigations of pollen tubes. The system permits large-scale germination, growth, and optical phenotyping of pollen tubes empowering rapid micro-indentation measurements on these cells.