Dual photonic bandgap hollow sphere colloidal photonic crystals for real-time fluorescence enhancement in living cells.

To overcome the problems of refractive index matching and increased disorder when working with traditional heterostructure colloidal photonic crystals (CPCs) with dual or multiple photonic bandgaps (PBGs) for fluorescence enhancement in water, we propose the use of a chemical heterostructure in hollow sphere CPCs (HSCPCs). A partial chemical modification of the HSCPC creates a large contrast in wettability to induce the heterostructure, while the hollow spheres increase the refractive index difference when used in aqueous environment. With the platform, fluorescence enhancement reaches around 160 times in solution, and 72 times (signal-to-background ratio ~7 times) in cells during proof-of-concept live cardiomyocyte contractility experiments. Such photonic platform can be further exploited for chemical sensing, bioassays, and environmental monitoring. Moreover, the introduction of chemical heterostructures provides new design principles for functionalized photonic devices.

High-Density Electrical Recording and Impedance Imaging With a Multi-Modal CMOS Multi-Electrode Array Chip.

Multi-electrode arrays, both active or passive, emerged as ideal technologies to unveil intricated electrophysiological dynamics of cells and tissues. Active MEAs, designed using complementary metal oxide semiconductor technology (CMOS), stand over passive devices thanks to the possibility of achieving single-cell resolution, the reduced electrode size, the reduced crosstalk and the higher functionality and portability. Nevertheless, most of the reported CMOS MEA systems mainly rely on a single operational modality, which strongly hampers the applicability range of a single device. This can be a limiting factor considering that most biological and electrophysiological dynamics are often based on the synergy of multiple and complex mechanisms acting from different angles on the same phenomena. Here, we designed a CMOS MEA chip with 16,384 titanium nitride electrodes, 6 independent operational modalities and 1,024 parallel recording channels for neuro-electrophysiological studies. Sixteen independent active areas are patterned on the chip surface forming a 4 × 4 matrix, each one including 1,024 electrodes. Electrodes of four different sizes are present on the chip surface, ranging from 2.5 × 3.5 µm2 up to 11 × 11.0 µm2, with 15 µm pitch. In this paper, we exploited the impedance monitoring and voltage recording modalities not only to monitor the growth and development of primary rat hippocampal neurons, but also to assess their electrophysiological activity over time showing a mean spike amplitude of 144.8 ± 84.6 µV. Fixed frequency (1 kHz) and high sampling rate (30 kHz) impedance measurements were used to evaluate the cellular adhesion and growth on the chip surface. Thanks to the high-density configuration of the electrodes, as well as their dimension and pitch, the chip can appreciate the evolutions of the cell culture morphology starting from the moment of the seeding up to mature culture conditions. The measurements were confirmed by fluorescent staining. The effect of the different electrode sizes on the spike amplitudes and noise were also discussed. The multi-modality of the presented CMOS MEA allows for the simultaneous assessment of different physiological properties of the cultured neurons. Therefore, it can pave the way both to answer complex fundamental neuroscience questions as well as to aid the current drug-development paradigm.

Action potential-based MEA platform for in vitro screening of drug-induced cardiotoxicity using human iPSCs and rat neonatal myocytes.

Drug-induced cardiotoxicity poses a negative impact on public health and drug development. Cardiac safety pharmacology issues urged for the preclinical assessment of drug-induced ventricular arrhythmia leading to the design of several in vitro electrophysiological screening assays. In general, patch clamp systems allow for intracellular recordings, while multi-electrode array (MEA) technology detect extracellular activity. Here, we demonstrate a complementary metal oxide semiconductor (CMOS)-based MEA system as a reliable platform for non-invasive, long-term intracellular recording of cardiac action potentials at high resolution. Quinidine (8 concentrations from 10-7 to 2.10-5M) and verapamil (7 concentrations from 10-11 to 10-5M) were tested for dose-dependent responses in a network of cardiomyocytes. Electrophysiological parameters, such as the action potential duration (APD), rates of depolarization and repolarization and beating frequency were assessed. In hiPSC, quinidine prolonged APD with EC50 of 2.2·10-6M. Further analysis indicated a multifactorial action potential prolongation by quinidine: (1) decreasing fast repolarization with IC50 of 1.1·10-6M; (2) reducing maximum upstroke velocity with IC50 of 2.6·10-6M; and (3) suppressing spontaneous activity with EC50 of 3.8·10-6M. In rat neonatal cardiomyocytes, verapamil blocked spontaneous activity with EC50 of 5.3·10-8M and prolonged the APD with EC50 of 2.5·10-8M. Verapamil reduced rates of fast depolarization and repolarization with IC50s of 1.8 and 2.2·10-7M, respectively. In conclusion, the proposed action potential-based MEA platform offers high quality and stable long-term recordings with high information content allowing to characterize multi-ion channel blocking drugs. We anticipate application of the system as a screening platform to efficiently and cost-effectively test drugs for cardiac safety.

Amyloid beta oligomers induce neuronal elasticity changes in age-dependent manner: a force spectroscopy study on living hippocampal neurons.

Small soluble species of amyloid-beta (Aß) formed during early peptide aggregation stages are responsible for several neurotoxic mechanisms relevant to the pathology of Alzheimer's disease (AD), although their interaction with the neuronal membrane is not completely understood. This study quantifies the changes in the neuronal membrane elasticity induced by treatment with the two most common Aß isoforms found in AD brains: Aß40 and Aß42. Using quantitative atomic force microscopy (AFM), we measured for the first time the static elastic modulus of living primary hippocampal neurons treated with pre-aggregated Aß40 and Aß42 soluble species. Our AFM results demonstrate changes in the elasticity of young, mature and aged neurons treated for a short time with the two Aß species pre-aggregated for 2 hours. Neurons aging under stress conditions, showing aging hallmarks, are the most susceptible to amyloid binding and show the largest decrease in membrane stiffness upon Aß treatment. Membrane stiffness defines the way in which cells respond to mechanical forces in their environment and has been shown to be important for processes such as gene expression, ion-channel gating and neurotransmitter vesicle transport. Thus, one can expect that changes in neuronal membrane elasticity might directly induce functional changes related to neurodegeneration.

Fabrication and successful in-vivo implantation of a flexible neural implant with a hybrid polyimide-silicon design.

A flexible neural implant was designed and fabricated using an novel integration approach that offers the advantages of both silicon and polymer based implants: high density electrodes and precise insertion on one side and mechanical flexibility suitable for reduced tissue strain due to micro-motion during chronic implantation on the other side. This was achieved by separating the device into silicon or polymer areas, depending on their desired functionality. The tip, where the recording and stimulation electrodes would be placed, was kept of silicon: a choice that doesn't call for any compromise to be made regarding the high density electrode and possible local circuit integration later on. The bevel shaped sharp silicon tip also proved to facilitate the probe insertion, offering a behavior very much similar to the classical rigid silicon probes. On the other side, most of the 1 cm long shank of the probe was made out of polyimide. This led to more than one order of magnitude reduction of the forces necessary to bend the shank. The flexible shank proved also to be more robust than silicon probes, sustaining significant deformation in any direction without fracture. The 9mm deep in-vivo implantation were successfully achieved without buckling for 10 µm/s and 100 µm/s insertion speeds.

Towards a closed-loop system for stimulation and recording: an in vitro approach with embryonic cardiomyocytes.

Closed loop systems, in which stimulation parameters are adjusted according to recorded signals would reduce the occurrence of side effects of stimulation and broaden current therapeutic options. As a step towards a closed-loop clinical system, we developed a single electrode stimulation / recording system for an in vitro microelectrode array. The system was used in vitro to simultaneously stimulate and record cardiac cells. Results indicated that stimulation artifacts depend on the distance between recording electrode and stimulating electrode and on the voltage amplitude. No artifact reduction algorithm was required for detecting cardiac action potentials 2ms after stimulation if the stimulation pulses were less than or equal to ± 1.5 V, and the distance from stimulation site was more than 200 µm. Cardiac signal propagation was also investigated with this system.

MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain: validation of various MPIO labeling strategies.

The adult rodent brain contains neural progenitor cells (NPCs), generated in the subventricular zone (SVZ), which migrate along the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they differentiate into neurons. The aim of this study was to visualize endogenous NPC migration along the RMS with magnetic resonance imaging (MRI) in adult healthy mice. We evaluated various in situ (in vivo) labeling approaches using micron-sized iron oxide particles (MPIOs) on their efficiency to label endogenous NPCs. In situ labeling and visualization of migrating NPCs were analyzed by a longitudinal MRI study and validated with histology. Here, we visualized endogenous NPC migration in the mouse brain by in vivo MRI and demonstrated accumulation of MPIO-labeled NPCs in the OB over time with ex vivo MRI. Furthermore, we investigated the influence of in situ injection of MPIOs on adult neurogenesis. Quantitative analysis of bromodeoxyuridine labeled cells revealed altered proliferation in the SVZ and NPC migration after in situ MPIO injection. From the labeling strategies presented in this report, intraventricular injection of a small number of MPIOs combined with the transfection agent poly-l-lysine hydrobromide was the best method as labeling of the NPCs was successful and proliferation in the SVZ was only marginally affected. While MRI visualization of endogenous NPC migration can provide insight into aberrant NPC migration in disease models, this work emphasizes the importance to carefully explore the impact on adult neurogenesis when new in situ labeling strategies are developed.