Miniaturized engineered heart tissues from hiPSC-derived triple cell type co-cultures to study human cardiac function.

Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better than standard two-dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 × 104 cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery.

Quantum Signature of a Squeezed Mechanical Oscillator.

Recent optomechanical experiments have observed nonclassical properties in macroscopic mechanical oscillators. A key indicator of such properties is the asymmetry in the strength of the motional sidebands produced in the probe electromagnetic field, which is originated by the noncommutativity between the oscillator ladder operators. Here we extend the analysis to a squeezed state of an oscillator embedded in an optical cavity, produced by the parametric effect originated by a suitable combination of optical fields. The motional sidebands assume a peculiar shape, related to the modified system dynamics, with asymmetric features revealing and quantifying the quantum component of the squeezed oscillator motion.

Fabrication and characterization of polyimide-based 'smooth' titanium nitride microelectrode arrays for neural stimulation and recording.

OBJECTIVE: As electrodes are required to interact with sub-millimeter neural structures, innovative microfabrication processes are required to enable fabrication of microdevices involved in such stimulation and/or recording. This requires the development of highly integrated and miniaturized systems, comprising die-integration-compatible technology and flexible microelectrodes. To elicit selective stimulation and recordings of sub-neural structures, such microfabrication process flow can beneficiate from the integration of titanium nitride (TiN) microelectrodes onto a polyimide substrate. Finally, assembling onto cuffs is required, as well as electrode characterization. APPROACH: Flexible TiN microelectrode array integration and miniaturization was achieved through microfabrication technology based on microelectromechanical systems (MEMS) and complementary metal-oxide semiconductor processing techniques and materials. They are highly reproducible processes, granting extreme control over the feature size and shape, as well as enabling the integration of on-chip electronics. This design is intended to enhance the integration of future electronic modules, with high gains on device miniaturization. MAIN RESULTS: (a) Fabrication of two electrode designs, (1) 2 mm long array with 14 TiN square-shaped microelectrodes (80 × 80 µm2), and (2) an electrode array with 2 mm × 80 µm contacts. The average impedances at 1 kHz were 59 and 5.5 kΩ, respectively, for the smaller and larger contacts. Both designs were patterned on a flexible substrate and directly interconnected with a silicon chip. (b) Integration of flexible microelectrode array onto a cuff electrode designed for acute stimulation of the sub-millimeter nerves. (c) The TiN electrodes exhibited capacitive charge transfer, a water window of -0.6 V to 0.8 V, and a maximum charge injection capacity of 154 ± 16 µC cm-2. SIGNIFICANCE: We present the concept, fabrication and characterization of composite and flexible cuff electrodes, compatible with post-processing and MEMS packaging technologies, which allow for compact integration with control, readout and RF electronics. The fabricated TiN microelectrodes were electrochemically characterized and exhibited a comparable performance to other state-of-the-art electrodes for neural stimulation and recording. Therefore, the presented TiN-on-polyimide microelectrodes, released from silicon wafers, are a promising solution for neural interfaces targeted at sub-millimeter nerves, which may benefit from future upgrades with die-electronic modules.

Microfabricated tuneable and transferable porous PDMS membranes for Organs-on-Chips.

We present a novel and highly reproducible process to fabricate transferable porous PDMS membranes for PDMS-based Organs-on-Chips (OOCs) using microelectromechanical systems (MEMS) fabrication technologies. Porous PDMS membranes with pore sizes down to 2.0 µm in diameter and a wide porosity range (2-65%) can be fabricated. To overcome issues normally faced when using replica moulding and extend the applicability to most OOCs and improve their scalability and reproducibility, the process includes a sacrificial layer to easily transfer the membranes from a silicon carrier to any PDMS-based OOC. The highly reliable fabrication and transfer method does not need of manual handling to define the pore features (size, distribution), allowing very thin (<10 µm) functional membranes to be transferred at chip level with a high success rate (85%). The viability of cell culturing on the porous membranes was assessed by culturing two different cell types on transferred membranes in two different OOCs. Human umbilical endothelial cells (HUVEC) and MDA-MB-231 (MDA) cells were successfully cultured confirming the viability of cell culturing and the biocompatibility of the membranes. The results demonstrate the potential of controlling the porous membrane features to study cell mechanisms such as transmigrations, monolayer formation, and barrier function. The high control over the membrane characteristics might consequently allow to intentionally trigger or prevent certain cellular responses or mechanisms when studying human physiology and pathology using OOCs.

Ultra-thin alumina and silicon nitride MEMS fabricated membranes for the electron multiplication.

In this paper we demonstrate the fabrication of large arrays of ultrathin freestanding membranes (tynodes) for application in a timed photon counter (TiPC), a novel photomultiplier for single electron detection. Low pressure chemical vapour deposited silicon nitride (Si x N y ) and atomic layer deposited alumina (Al2O3) with thicknesses down to only 5 nm are employed for the membrane fabrication. Detailed characterization of structural, mechanical and chemical properties of the utilized films is carried out for different process conditions and thicknesses. Furthermore, the performance of the tynodes is investigated in terms of secondary electron emission, a fundamental attribute that determines their applicability in TiPC. Studied features and presented fabrication methods may be of interest for other MEMS application of alumina and silicon nitride as well, in particular where strong ultra-thin membranes are required.

Dynamical Two-Mode Squeezing of Thermal Fluctuations in a Cavity Optomechanical System.

We report the experimental observation of two-mode squeezing in the oscillation quadratures of a thermal micro-oscillator. This effect is obtained by parametric modulation of the optical spring in a cavity optomechanical system. In addition to stationary variance measurements, we describe the dynamic behavior in the regime of pulsed parametric excitation, showing an enhanced squeezing effect surpassing the stationary 3 dB limit. While the present experiment is in the classical regime, our technique can be exploited to produce entangled, macroscopic quantum optomechanical modes.

Mixed motion in deterministic ratchets due to anisotropic permeability.

Nowadays microfluidic devices are becoming popular for cell/DNA sorting and fractionation. One class of these devices, namely deterministic ratchets, seems most promising for continuous fractionation applications of suspensions (Kulrattanarak et al., 2008 [1]). Next to the two main types of particle behavior, zigzag and displacement motion as noted by the inventors (Huang et al., 2004 [2]) and (Inglis et al., 2006 [3]), we have shown recently the existence of a intermediate particle behavior, which we named 'mixed motion'. In this paper we formulate the hypothesis that the occurrence of mixed motion is correlated with anisotropy in the permeability of the obstacle array. This hypothesis we base on the comparison of experimental observations of mixed motion and the flow lane distribution as obtained from 2-D flow simulations.

Atomic-scale electron microscopy at ambient pressure.

We demonstrate a novel nanoreactor for performing atomic-resolution environmental transmission electron microscopy (ETEM) of nanostructured materials during exposure to gases at ambient pressures and elevated temperatures. The nanoreactor is a microelectromechanical system (MEMS) and is functionalized with a micrometer-sized gas-flow channel, electron-transparent windows and a heating device. It fits into the tip of a dedicated sample holder that can be used in a normal CM microscope of Philips/FEI Company. The nanoreactor performance was demonstrated by ETEM imaging of a Cu/ZnO catalyst for methanol synthesis during exposure to hydrogen. Specifically, the nanoreactor facilitated the direct observation of Cu nanocrystal growth and mobility on a sub-second time scale during heating to 500 degrees C and exposure to 1.2 bar of H(2). For the same gas reaction environment, ETEM images show atomic lattice fringes in the Cu nanocrystals with spacing of 0.18 nm, attesting the spatial resolution limit of the system. The nanoreactor concept opens up new possibilities for in situ studies of nanomaterials and the ways they interact with their ambient working environment in diverse areas, such as heterogeneous catalysis, electrochemistry, nanofabrication, materials science and biology.

Development and characterization of an integrated silicon micro flow cytometer.

This paper describes an innovative integrated micro flow cytometer that presents a new arrangement for the excitation/detection system. The sample liquid, containing the fluorescent marked particles/cells under analysis, is hydrodynamically squeezed into a narrow stream by two sheath flows so that the particles/cells flow individually through a detection region. The detection of the particles/cells emitted fluorescence is carried out by using a collection fiber placed orthogonally to the flow. The device is based on silicon hollow core antiresonant reflecting optical waveguides (ARROWs). ARROW geometry allows one to use the same channel to guide both the sample stream and the fluorescence excitation light, leading to a simplification of the optical configuration and to an increase of the signal-to-noise ratio. The integrated micro flow cytometer has been characterized by using biological samples marked with standard fluorochromes. The experimental investigation confirms the success of the proposed microdevice in the detection of cells.

Single-mask microfabrication of aspherical optics using KOH anisotropic etching of Si.

We report on the microfabrication of continuous aspherical optical surfaces with a single-mask process, using anisotropic etching of silicon in a KOH water solution. Precise arbitrary aspherical surfaces with lateral scales on the order of several millimeters and a profile depth on the order of several micrometers were fabricated using this process. We discuss the factors defining the precision of the formed component and the resulting surface quality. We demonstrate 1 mm and 5 mm replicated aspherical phase plates, reproducing defocus, tilt, astigmatism and high-order aberrations. The technology has a potential for serial production of reflective and refractive arbitrary aspherical micro-optical components.

Integrated flow-cells for novel adjustable sheath flows.

In this paper two integrated flow-cells are presented that can generate novel sheath flows. The flow-cells allow for dynamic orthogonal control of the sample flow dimensions. In addition to this, the sample flow can be freely positioned inside the channel. The flow-cells are attractive, because they are very simple to fabricate and are compatible with the integration of sensors. Experiments have been carried out demonstrating that the sample flow dimensions can be controlled over a wide range; also the results show good agreement with finite element simulation results.

Flexible mirror micromachined in silicon.

An electrostatically controlled flexible mirror has been fabricated on a silicon chip by means of bulk micromachining. The mirror has a 10.5 mm × 10.5 mm square aperture and consists of a 0.5-µm-thick tensile-stressed silicon-nitride diaphragm coated with a 0.2-µm-thick reflective aluminum layer. The reflecting surface is initially plane with a mean-square deviation of ~λ/8 for λ = 633 nm. The shape of the reflecting surface is controlled electrostatically by an array of integrated actuators. Good initial optical quality and the possibility of electrostatic control of the reflecting surface make the on-chip mirror useful for various electro-optical applications.