As a result of current trends in miniaturisation and the need for faster electronic circuits, integrated circuit (IC) design has become more complex. Closely packed conductors carrying radiofrequency (RF) signals are subjected to parasitic coupling, complicating the IC design and validation. Prototyping of such devices is supported by contact probes that make an ohmic connection to contact pads. These pads take up valuable space and may interfere with the design. Alternatively, near-field probing techniques, that utilise capacitive and inductive coupling, have been employed for local RF voltage and current characterisation. In this research, such a near-field probe is developed through a multiscale 3D-printing process. It contains a miniaturised conductive loop, enclosing an area of 12.5µm2, and a conductive tip with an apex radius of 100nm. A model-based approach, that makes use of the discrepancy between parasitic long-range, and local short-range contributions to the measurement signal, is expanded to conduct both RF voltage and current measurements with increased spatial resolution. With this approach, combined contactless RF-voltage and RF-current measurements were executed demonstrating a measurement bandwidth from 1 to 23GHz. Capacitively coupled RF-voltage measurements achieved a spatial resolution of 8µm, while the spatial resolution of inductively coupled current measurements was only simulated. The simulation shows an expected spatial resolution of 3µm.

Cryogenic electron microscopy (cryoEM) has become an indispensable technique for determining the structures of isolated biological macromolecules and imaging biomolecular structures within cells. While thin films of frozen-hydrated macromolecule suspensions can be directly prepared and imaged, these molecules must be extracted from their cellular context using existing sample preparation methods. On the other hand, cellular specimens preserve the native context, but the region of interest must be sectioned or subjected to focused ion-beam milling at cryogenic temperatures to achieve the necessary thickness for transmission electron imaging. Currently, no method exists for targeted cytoplasmic extraction of a subcellular volume from individual cells for direct vitrification and cryoEM imaging. In this study, a method is presented that addresses this gap. A system has developed that utilizes a force-sensitive microfluidic cantilever pipette to aspirate and dispense sample volumes as small as 204 fL onto conventional cryoEM sample supports, maintained at the dew point. This is followed by automatic vitrification for cryoEM imaging. Coupled with a fluorescence microscope, this setup allows for the extraction of a targeted subcellular volume from an individual cell and subsequent dispensing of the aspirated content onto an electron microscopy grid. A proof-of-concept is demonstrated by dispensing femtolitre volumes of the standard cryoEM single-particle sample, tobacco mosaic virus, and performing a subcellular biopsy from a single HeLa cell. Additionally, the challenges of manipulating such small volumes for cryoEM sample preparation are discussed, highlighting the current limitations of this approach and potential solutions for overcoming them.

The Poisson's ratio and elastic modulus are two parameters determining the elastic behavior of biomaterials. While the effects of elastic modulus on the cell response is widely studied, very little is known regarding the effects of the Poisson's ratio. The micro-architecture of meta-biomaterials determines not only the Poisson's ratio but also several other parameters that also influence cell response, such as porosity, pore size, and effective elastic modulus. It is, therefore, very challenging to isolate the effects of the Poisson's ratio from those of other micro-architectural parameters. Here, we computationally design meta-biomaterials with controlled Poisson's ratios, ranging between -0.74 and +0.74, while maintaining consistent porosity, pore size, and effective elastic modulus. The 3D meta-biomaterials were additively manufactured at the micro-scale using two-photon polymerization (2PP), and were mechanically evaluated at the meso‑scale. The response of murine preosteoblasts to these meta-biomaterials was then studied using in vitro cell culture models. Meta-biomaterials with positive Poisson's ratios resulted in higher metabolic activity than those with negative values. The cells could attach and infiltrate all meta-biomaterials from the bottom to the top, fully covering the scaffolds after 17 days of culture. Interestingly, the meta-biomaterials exhibited different cell-induced deformations (e.g., shrinkage or local bending) as observed via scanning electron microscopy. The outcomes of osteogenic differentiation (i.e., Runx2 immunofluorescent staining) and matrix mineralization (i.e., Alizarin red staining) assays indicated the significant potential impact of these meta-biomaterials in the field of bone tissue engineering, paving the way for the development of advanced bone meta-implants. Statement of significance: We studied the influence of Poisson's ratio on bone cell response in meta-biomaterials. While elastic modulus effects are well-studied, the impact of Poisson's ratio, especially negative values found in architected biomaterials, remains largely unexplored. The complexity arises from intertwined micro-architectural parameters, such as porosity and elastic modulus, making it challenging to isolate the Poisson's ratio. To overcome this limitation, this study employed rational computational design to create meta-biomaterials with controlled Poisson's ratios, alongside consistent effective elastic modulus, porosity, and pore size. The study reveals that two-photon polymerized 3D meta-biomaterials with positive Poisson's ratios displayed higher metabolic activity, while all the developed meta-biomaterials supported osteogenic differentiation of preosteoblasts as well as matrix mineralization. The outcomes pave the way for the development of advanced 3D bone tissue models and meta-implants.

Mechanical and morphological design parameters, such as stiffness or porosity, play important roles in creating orthopedic implants and bone substitutes. However, we have only a limited understanding of how the microarchitecture of porous scaffolds contributes to bone regeneration. Meta-biomaterials are increasingly used to precisely engineer the internal geometry of porous scaffolds and independently tailor their mechanical properties (e.g., stiffness and Poisson's ratio). This is motivated by the rare or unprecedented properties of meta-biomaterials, such as negative Poisson's ratios (i.e., auxeticity). It is, however, not clear how these unusual properties can modulate the interactions of meta-biomaterials with living cells and whether they can facilitate bone tissue engineering under static and dynamic cell culture and mechanical loading conditions. Here, we review the recent studies investigating the effects of the Poisson's ratio on the performance of meta-biomaterials with an emphasis on the relevant mechanobiological aspects. We also highlight the state-of-the-art additive manufacturing techniques employed to create meta-biomaterials, particularly at the micrometer scale. Finally, we provide future perspectives, particularly for the design of the next generation of meta-biomaterials featuring dynamic properties (e.g., those made through 4D printing).

Quantifying the nanomechanical properties of soft-matter using multi-frequency atomic force microscopy (AFM) is crucial for studying the performance of polymers, ultra-thin coatings, and biological systems. Such characterization processes often make use of cantilever's spectral components to discern nanomechanical properties within a multi-parameter optimization problem. This could inadvertently lead to an over-determined parameter estimation with no clear relation between the identified parameters and their influence on the experimental data. In this work, we explore the sensitivity of viscoelastic characterization in polymeric samples to the experimental observables of multi-frequency intermodulation AFM. By performing simulations and experiments we show that surface viscoelasticity has negligible effect on the experimental data and can lead to inconsistent and often non-physical identified parameters. Our analysis reveals that this lack of influence of the surface parameters relates to a vanishing gradient and non-convexity while minimizing the objective function. By removing the surface dependency from the model, we show that the characterization of bulk properties can be achieved with ease and without any ambiguity. Our work sheds light on the sensitivity issues that can be faced when optimizing for a large number of parameters and observables in AFM operation, and calls for the development of new viscoelastic models at the nanoscale and improved computational methodologies for nanoscale mapping of viscoelasticity using AFM.

Increasing the signal-to-noise ratio in dynamic atomic force microscopy plays a key role in nanomechanical mapping of materials with atomic resolution. In this work, we develop an experimental procedure for increasing the sensitivity of higher harmonics of an atomic-force-microscope cantilever without modifying the cantilever geometry but instead by utilizing dynamical mode coupling between its flexural modes of vibration. We perform experiments on different cantilevers and samples and observe that via nonlinear resonance frequency tuning we can obtain a frequency range where strong modal interactions lead to 7-fold and 16-fold increases in the sensitivity of the 6th and 17th harmonics while reducing sample indentation. We derive a numerical model that captures the observed physics and confirms that nonlinear mode coupling is the reason for the increase of the amplitude of higher harmonics during tip-sample interactions.

Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.

There is a high demand for novel flexible micro-devices for energy harvesting from low-frequency and random mechanical sources. The research of new functional designs is required to strategically enhance the performances and to increase the control on mechanical flexibility. In this work we report the fabrication and characterization of bi-stable and statically balanced thin-film piezoelectric transducers based on Aluminum Nitride (AlN). The device consists of a piezoelectric layer sandwiched between two thin Molybdenum electrodes that were deposited on a Kapton substrate by reactive sputtering and patterned by UV lithography. In order to improve the out-of-plane flexibility, the mechanical design is distinguished by a post-buckled flexure that introduces a negative stiffness to compensate the otherwise positive stiffness of the system. The buckling was introduced by a new method, called Package-Induced Preloading (PIP) where the mechanisms are laminated over a package with a geometry extending out-of-plane. The induced buckling resulted in bi-stable and statically balanced mechanisms which demonstrated an enhanced voltage output during a triggered snapping step. A preliminary study shows potential for the statically balanced designs and the PIP method for wind energy harvesting, revealing prospective applications and future improvements for the development of energy harvesters.

Advances in nanofabrication over the past twenty years have enabled the creation and use of ever-more interesting and useful micromachines. Optical micromachines are a particularly attractive subset of these for researchers in biological and soft-matter sciences, due to their potential to aid in optical tweezer studies of laser-sensitive samples. However, the development of multi-component micromachines is made difficult due to the dominance of surface forces at this scale, which is made all the more relevant in the high-salt concentrations used for biological studies. This study concerns the design of simple, first-class lever micromachines for use in environments with different salt concentrations, in an attempt to provide a guideline for design requirements of functional optical micromachines for use in physiological conditions.

Organ-on-chip (OoC) technology is increasingly used for biomedical research and to speed up the process of bringing a drug from lab to the market. The main fluidic components of an OoC device are microfluidic channels and porous membranes arranged in three dimensions. Current chips are often assembled from several parts. In the development phase a small change in design will cause a delay in the research because a new prototype has to be built and assembled again step-by-step. The research discussed in this paper addresses this point by targeting a monolithic 3D device that can be fabricated in a single lithography and development step, enabling rapid prototyping. Two-photon lithography (TPL) was used in combination with a positive photoresist AZ 4562. The exposure process was characterized, which included an experimental and theoretical study of the voxel size and shape. It was found that the voxel has an hourglass-shape for the laser power settings that were required for process stability. The smallest pores we could produce with these settings measured 250 nm in diameter. The TPL process was then used to fabricate a microfluidic device featuring two crossed channels each one on a separate height-level, connected by a membrane in the centre. Access to the channels was provided through 4 reservoirs from the top-side of the device. The device was successfully filled with water and dried to see whether it can withstand the corresponding capillary forces.

We introduce a two-channel microfluidic atomic force microscopy (AFM) cantilever that combines the nanomechanical sensing functionality of an AFM cantilever with the ability to manipulate fluids of picolitres or smaller volumes through nanoscale apertures near the cantilever tip. Each channel is connected to a separate fluid reservoir, which can be independently controlled by pressure. Various systematic experiments with fluorescent liquids were done by either injecting the liquids from the on-chip reservoir or aspirating directly through the nanoscale apertures at the tip. A flow rate analysis of volume dosing, aspiration and concentration dosing inside the liquid medium was performed. To understand the fluid behaviour, an analytical model based on the hydrodynamic resistance, as well as numerical flow simulations of single and multi-phase conditions were performed and compared. By applying pressures between -500 mbar and 500 mbar to the reservoirs of the probe with respect to the ambient pressure, flow rates ranging from 10 fl s-1 to 83 pl s-1 were obtained inside the channels of the cantilever as predicted by the analytical model. The smallest dosing flow rate through the apertures was 720 fl s-1, which was obtained with a 10 mbar pressure on one reservoir and ambient pressure on the other. The solute concentration in the outflow could be tuned to values between 0% and 100% by pure convection and to values between 17.5% and 90% in combination with diffusion. The results prove that this new probe enables handling multiple fluids with the scope to inject different concentrations of analytes inside a single living cell and also perform regular AFM functionalities.