Microelectrode arrays of diamond-insulated graphitic channels for real time detection of exocytotic events from cultured chromaffin cells and slices of adrenal glands

A microstructured graphitic 4x4 multielectrode array was embedded in a single crystal diamond substrate (4x4 {uG-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time effective fabrication of extended arrays with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20x3.5 um^2) separated by 200 um gaps. Taking advantage of the array geometry we addressed the following specific issues: i) detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, ii) resolve the waveform of different subsets of exocytotic events, iii) monitoring quantal secretory events from thin slices of the adrenal gland. The frequency of spontaneous release was low (0.12 Hz and 0.3 Hz respectively for adrenal slices and cultured cells) and increased up to 0.9 Hz after stimulation with 30 mM KCl in cultured cells. The spike amplitude as well as rise and decay time were comparable with those measured by carbon fiber microelectrodes and allowed to identify three different subsets of secretory events associated to "full fusion" events, "kiss and-run" and "kiss-and-stay" exocytosis, confirming that the device has adequate sensitivity and time resolution for real-time recordings. The device offers the significant advantage of shortening the time to collect data by allowing simultaneous recordings from cell populations either in primary cell cultures or in intact tissues

Kelvin probe characterization of buried graphitic microchannels in single-crystal diamond

In this work, we present an investigation by Kelvin Probe Microscopy (KPM) of buried graphitic microchannels fabricated in single-crystal diamond by direct MeV ion microbeam writing. Metal deposition of variable-thickness masks was adopted to implant channels with emerging endpoints and high temperature annealing was performed in order to induce the graphitization of the highly-damaged buried region. When an electrical current was flowing through the biased buried channel, the structure was clearly evidenced by KPM maps of the electrical potential of the surface region overlying the channel at increasing distances from the grounded electrode. The KPM profiling shows regions of opposite contrast located at different distances from the endpoints of the channel. This effect is attributed to the different electrical conduction properties of the surface and of the buried graphitic layer. The model adopted to interpret these KPM maps and profiles proved to be suitable for the electronic characterization of buried conductive channels, providing a non-invasive method to measure the local resistivity with a micrometer resolution. The results demonstrate the potential of the technique as a powerful diagnostic tool to monitor the functionality of all-carbon graphite/diamond devices to be fabricated by MeV ion beam lithography.Comment: 21 pages, 5 figure

IMAGE QUALITY IMPROVEMENT BY ADAPTIVE EXPOSURE CORRECTION TECHNIQUES

The proposed paper concerns the processing of images in digital format and, more specifically, particular techniques that can be advantageously used in digital still cameras for improving the quality of images acquired with a non-optimal exposure. The proposed approach analyses the CCD/CMOS sensor Bayer data or the corresponding color generated image and, after identifying specific features, it adjusts the exposure level according to a ‘camera response ’ like function. 1

Laser-induced persistent photovoltage on the surface of a ternary topological insulator at room temperature

Using time- and angle-resolved photoemission, we investigate the ultrafast response of excited electrons in the ternary topological insulator (Bi$_{1 x}$Sb$_{x}$)$_2$Te$_3$ to fs-infrared pulses. We demonstrate that at the critical concentration $x$=0.55, where the system becomes bulk insulating, a surface voltage can be driven at room temperature through the topological surface state solely by optical means. We further show that such a photovoltage persists over a time scale that exceeds $\sim$6 $\mu$s, i.e, much longer than the characteristic relaxation times of bulk states. We attribute the origin of the photovoltage to a laser-induced band-bending effect which emerges near the surface region on ultrafast time scales. The photovoltage is also accompanied by a remarkable increase in the relaxation times of excited states as compared to undoped topological insulators. Our findings are relevant in the context of applications of topological surface states in future optical devices.Comment: 5 pages, 4 figure

All-carbon multi-electrode array for real-time in vitro measurements of oxidizable neurotransmitters

We report on the ion beam fabrication of all-carbon multi electrode arrays (MEAs) based on 16 graphitic micro-channels embedded in single-crystal diamond (SCD) substrates. The fabricated SCD-MEAs are systematically employed for the in vitro simultaneous amperometric detection of the secretory activity from populations of chromaffin cells, demonstrating a new sensing approach with respect to standard techniques. The biochemical stability and biocompatibility of the SCD-based device combined with the parallel recording of multi-electrodes array allow: i) a significant time saving in data collection during drug screening and/or pharmacological tests over a large number of cells, ii) the possibility of comparing altered cell functionality among cell populations, and iii) the repeatition of acquisition runs over many cycles with a fully non-toxic and chemically robust bio-sensitive substrate.Comment: 24 pages, 5 figure

Nanodiamonds-induced effects on neuronal firing of mouse hippocampal microcircuits

Fluorescent nanodiamonds (FND) are carbon-based nanomaterials that can efficiently incorporate optically active photoluminescent centers such as the nitrogen-vacancy complex, thus making them promising candidates as optical biolabels and drug-delivery agents. FNDs exhibit bright fluorescence without photobleaching combined with high uptake rate and low cytotoxicity. Focusing on FNDs interference with neuronal function, here we examined their effect on cultured hippocampal neurons, monitoring the whole network development as well as the electrophysiological properties of single neurons. We observed that FNDs drastically decreased the frequency of inhibitory (from 1.81 Hz to 0.86 Hz) and excitatory (from 1.61 Hz to 0.68 Hz) miniature postsynaptic currents, and consistently reduced action potential (AP) firing frequency (by 36%), as measured by microelectrode arrays. On the contrary, bursts synchronization was preserved, as well as the amplitude of spontaneous inhibitory and excitatory events. Current-clamp recordings revealed that the ratio of neurons responding with AP trains of high-frequency (fast-spiking) versus neurons responding with trains of low-frequency (slow-spiking) was unaltered, suggesting that FNDs exerted a comparable action on neuronal subpopulations. At the single cell level, rapid onset of the somatic AP ("kink") was drastically reduced in FND-treated neurons, suggesting a reduced contribution of axonal and dendritic components while preserving neuronal excitability.Comment: 34 pages, 9 figure

Creation of pure non-crystalline diamond nanostructures via room-temperature ion irradiation and subsequent thermal annealing

Carbon exhibits a remarkable range of structural forms, due to the availability of sp3, sp2 and sp1 chemical bonds. Contrarily to other group IV elements such as silicon and germanium, the formation of an amorphous phase based exclusively on sp3 bonds is extremely challenging due to the strongly favored formation of graphitic-like structures at room 19 temperature and pressure. As such, the formation of a fully sp3-bonded carbon phase requires 20 an extremely careful (and largely unexplored) definition of the pressure and temperature across the phase diagram. Here, we report on the possibility of creating full-sp3 amorphous nanostructures within the bulk crystal of diamond with room-temperature ion-beam irradiation, followed by an annealing process that does not involve the application of any external mechanical pressure. As confirmed by numerical simulations, the (previously unreported) radiation-damage-induced formation of an amorphous sp2-free phase in diamond is determined by the buildup of extremely high internal stresses from the surrounding lattice, which (in the case of nanometer-scale regions) fully prevent the graphitization process. Besides the relevance of understanding the formation of exotic carbon phases, the use of focused/collimated ion beams discloses appealing perspectives for the direct fabrication of such nanostructures in complex three-dimensional geometries