HectoSTAR µLED Optoelectrodes for Large-Scale, High-Precision In Vivo Opto-Electrophysiology.

Dynamic interactions within and across brain areas underlie behavioral and cognitive functions. To understand the basis of these processes, the activities of distributed local circuits inside the brain of a behaving animal must be synchronously recorded while the inputs to these circuits are precisely manipulated. Even though recent technological advances have enabled such large-scale recording capabilities, the development of the high-spatiotemporal-resolution and large-scale modulation techniques to accompany those recordings has lagged. A novel neural probe is presented in this work that enables simultaneous electrical monitoring and optogenetic manipulation of deep neuronal circuits at large scales with a high spatiotemporal resolution. The "hectoSTAR" micro-light-emitting-diode (µLED) optoelectrode features 256 recording electrodes and 128 stimulation µLEDs monolithically integrated on the surface of its four 30-µm thick silicon micro-needle shanks, covering a large volume with 1.3-mm × 0.9-mm cross-sectional area located as deep as 6 mm inside the brain. The use of this device in behaving mice for dissecting long-distance network interactions across cortical layers and hippocampal regions is demonstrated. The recording-and-stimulation capabilities hectoSTAR µLED optoelectrodes enables will open up new possibilities for the cellular and circuit-based investigation of brain functions in behaving animals.

Artifact-free and high-temporal-resolution in vivo opto-electrophysiology with microLED optoelectrodes.

The combination of in vivo extracellular recording and genetic-engineering-assisted optical stimulation is a powerful tool for the study of neuronal circuits. Precise analysis of complex neural circuits requires high-density integration of multiple cellular-size light sources and recording electrodes. However, high-density integration inevitably introduces stimulation artifact. We present minimal-stimulation-artifact (miniSTAR) µLED optoelectrodes that enable effective elimination of stimulation artifact. A multi-metal-layer structure with a shielding layer effectively suppresses capacitive coupling of stimulation signals. A heavily boron-doped silicon substrate silences the photovoltaic effect induced from LED illumination. With transient stimulation pulse shaping, we reduced stimulation artifact on miniSTAR µLED optoelectrodes to below 50 µVpp, much smaller than a typical spike detection threshold, at optical stimulation of >50 mW mm-2 irradiance. We demonstrated high-temporal resolution (<1 ms) opto-electrophysiology without any artifact-induced signal quality degradation during in vivo experiments. MiniSTAR µLED optoelectrodes will facilitate functional mapping of local circuits and discoveries in the brain.

Dual color optogenetic control of neural populations using low-noise, multishank optoelectrodes.

Optogenetics allows for optical manipulation of neuronal activity and has been increasingly combined with intra- and extra-cellular electrophysiological recordings. Genetically-identified classes of neurons are optically manipulated, though the versatility of optogenetics would be increased if independent control of distinct neural populations could be achieved on a sufficient spatial and temporal resolution. We report a scalable multi-site optoelectrode design that allows simultaneous optogenetic control of two spatially intermingled neuronal populations in vivo. We describe the design, fabrication, and assembly of low-noise, multi-site/multi-color optoelectrodes. Each shank of the four-shank assembly is monolithically integrated with 8 recording sites and a dual-color waveguide mixer with a 7 × 30 µm cross-section, coupled to 405 nm and 635 nm injection laser diodes (ILDs) via gradient-index (GRIN) lenses to meet optical and thermal design requirements. To better understand noise on the recording channels generated during diode-based activation, we developed a lumped-circuit modeling approach for EMI coupling mechanisms and used it to limit artifacts to amplitudes under 100 µV upto an optical output power of 450 µW. We implanted the packaged devices into the CA1 pyramidal layer of awake mice, expressing Channelrhodopsin-2 in pyramidal cells and ChrimsonR in paravalbumin-expressing interneurons, and achieved optical excitation of each cell type using sub-mW illumination. We highlight the potential use of this technology for functional dissection of neural circuits.

State-of-the-art MEMS and microsystem tools for brain research.

Mapping brain activity has received growing worldwide interest because it is expected to improve disease treatment and allow for the development of important neuromorphic computational methods. MEMS and microsystems are expected to continue to offer new and exciting solutions to meet the need for high-density, high-fidelity neural interfaces. Herein, the state-of-the-art in recording and stimulation tools for brain research is reviewed, and some of the most significant technology trends shaping the field of neurotechnology are discussed.

Fiberless multicolor neural optoelectrode for in vivo circuit analysis.

Maximizing the potential of optogenetic approaches in deep brain structures of intact animals requires optical manipulation of neurons at high spatial and temporal resolutions, while simultaneously recording electrical data from those neurons. Here, we present the first fiber-less optoelectrode with a monolithically integrated optical waveguide mixer that can deliver multicolor light at a common waveguide port to achieve multicolor modulation of the same neuronal population in vivo. We demonstrate successful device implementation by achieving efficient coupling between a side-emitting injection laser diode (ILD) and a dielectric optical waveguide mixer via a gradient-index (GRIN) lens. The use of GRIN lenses attains several design features, including high optical coupling and thermal isolation between ILDs and waveguides. We validated the packaged devices in the intact brain of anesthetized mice co-expressing Channelrhodopsin-2 and Archaerhodopsin in pyramidal cells in the hippocampal CA1 region, achieving high quality recording, activation and silencing of the exact same neurons in a given local region. This fully-integrated approach demonstrates the spatial precision and scalability needed to enable independent activation and silencing of the same or different groups of neurons in dense brain regions while simultaneously recording from them, thus considerably advancing the capabilities of currently available optogenetic toolsets.

Chronic In Vivo Evaluation of PEDOT/CNT for Stable Neural Recordings.

OBJECTIVE: Subcellular-sized chronically implanted recording electrodes have demonstrated significant improvement in single unit (SU) yield over larger recording probes. Additional work expands on this initial success by combining the subcellular fiber-like lattice structures with the design space versatility of silicon microfabrication to further improve the signal-to-noise ratio, density of electrodes, and stability of recorded units over months to years. However, ultrasmall microelectrodes present very high impedance, which must be lowered for SU recordings. While poly(3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate (PSS) coating have demonstrated great success in acute to early-chronic studies for lowering the electrode impedance, concern exists over long-term stability. Here, we demonstrate a new blend of PEDOT doped with carboxyl functionalized multiwalled carbon nanotubes (CNTs), which shows dramatic improvement over the traditional PEDOT/PSS formula. METHODS: Lattice style subcellular electrode arrays were fabricated using previously established method. PEDOT was polymerized with carboxylic acid functionalized carbon nanotubes onto high-impedance (8.0 ± 0.1 MΩ: M ± S.E.) 250-µm(2) gold recording sites. RESULTS: PEDOT/CNT-coated subcellular electrodes demonstrated significant improvement in chronic spike recording stability over four months compared to PEDOT/PSS recording sites. CONCLUSION: These results demonstrate great promise for subcellular-sized recording and stimulation electrodes and long-term stability. SIGNIFICANCE: This project uses leading-edge biomaterials to develop chronic neural probes that are small (subcellular) with excellent electrical properties for stable long-term recordings. High-density ultrasmall electrodes combined with advanced electrode surface modification are likely to make significant contributions to the development of long-term (permanent), high quality, and selective neural interfaces.

Monolithically Integrated µLEDs on Silicon Neural Probes for High-Resolution Optogenetic Studies in Behaving Animals.

We report a scalable method to monolithically integrate microscopic light emitting diodes (µLEDs) and recording sites onto silicon neural probes for optogenetic applications in neuroscience. Each µLED and recording site has dimensions similar to a pyramidal neuron soma, providing confined emission and electrophysiological recording of action potentials and local field activity. We fabricated and implanted the four-shank probes, each integrated with 12 µLEDs and 32 recording sites, into the CA1 pyramidal layer of anesthetized and freely moving mice. Spikes were robustly induced by 60 nW light power, and fast population oscillations were induced at the microwatt range. To demonstrate the spatiotemporal precision of parallel stimulation and recording, we achieved independent control of distinct cells ∼ 50 µm apart and of differential somato-dendritic compartments of single neurons. The scalability and spatiotemporal resolution of this monolithic optogenetic tool provides versatility and precision for cellular-level circuit analysis in deep structures of intact, freely moving animals.

In vivo optical modulation of neural signals using monolithically integrated two-dimensional neural probe arrays.

Integration of stimulation modalities (e.g. electrical, optical, and chemical) on a large array of neural probes can enable an investigation of important underlying mechanisms of brain disorders that is not possible through neural recordings alone. Furthermore, it is important to achieve this integration of multiple functionalities in a compact structure to utilize a large number of the mouse models. Here we present a successful optical modulation of in vivo neural signals of a transgenic mouse through our compact 2D MEMS neural array (optrodes). Using a novel fabrication method that embeds a lower cladding layer in a silicon substrate, we achieved a thin silicon 2D optrode array that is capable of delivering light to multiple sites using SU-8 as a waveguide core. Without additional modification to the microelectrodes, the measured impedance of the multiple microelectrodes was below 1 MΩ at 1 kHz. In addition, with a low background noise level (± 25 µV), neural spikes from different individual neurons were recorded on each microelectrode. Lastly, we successfully used our optrodes to modulate the neural activity of a transgenic mouse through optical stimulation. These results demonstrate the functionality of the 2D optrode array and its potential as a next-generation tool for optogenetic applications.

Tools for probing local circuits: high-density silicon probes combined with optogenetics.

To understand how function arises from the interactions between neurons, it is necessary to use methods that allow the monitoring of brain activity at the single-neuron, single-spike level and the targeted manipulation of the diverse neuron types selectively in a closed-loop manner. Large-scale recordings of neuronal spiking combined with optogenetic perturbation of identified individual neurons has emerged as a suitable method for such tasks in behaving animals. To fully exploit the potential power of these methods, multiple steps of technical innovation are needed. We highlight the current state of the art in electrophysiological recording methods, combined with optogenetics, and discuss directions for progress. In addition, we point to areas where rapid development is in progress and discuss topics where near-term improvements are possible and needed.

An implantable neural probe with monolithically integrated dielectric waveguide and recording electrodes for optogenetics applications.

OBJECTIVE: Optogenetics promises exciting neuroscience research by offering optical stimulation of neurons with unprecedented temporal resolution, cell-type specificity and the ability to excite as well as to silence neurons. This work provides the technical solution to deliver light to local neurons and record neural potentials, facilitating local circuit analysis and bridging the gap between optogenetics and neurophysiology research. APPROACH: We have designed and obtained the first in vivo validation of a neural probe with monolithically integrated electrodes and waveguide. High spatial precision enables optical excitation of targeted neurons with minimal power and recording of single-units in dense cortical and subcortical regions. MAIN RESULTS: The total coupling and transmission loss through the dielectric waveguide at 473 nm was 10.5 ± 1.9 dB, corresponding to an average output intensity of 9400 mW mm(-2) when coupled to a 7 mW optical fiber. Spontaneous field potentials and spiking activities of multiple Channelrhodopsin-2 expressing neurons were recorded in the hippocampus CA1 region of an anesthetized rat. Blue light stimulation at intensity of 51 mW mm(-2) induced robust spiking activities in the physiologically identified local populations. SIGNIFICANCE: This minimally invasive, complete monolithic integration provides unmatched spatial precision and scalability for future optogenetics studies at deep brain regions with high neuronal density.

Demonstration of motionless Knudsen pump based micro-gas chromatography featuring micro-fabricated columns and on-column detectors.

This paper reports the investigation of a micro-gas chromatography (µGC) system that utilizes an array of miniaturized motionless Knudsen pumps (KPs) as well as microfabricated separation columns and optical detectors. A prototype system was built to achieve a flow rate of 1 mL min(-1) and 0.26 mL min(-1) for helium and dry air, respectively, when they were used as carrier gas. This system was then employed to evaluate GC performance compromises and demonstrate the ability to separate and detect gas mixtures containing analytes of different volatilities and polarities. Furthermore, the use of pressure programming of the KP array was demonstrated to significantly shorten the analysis time while maintaining a high detection resolution. Using this method, we obtained a high resolution detection of 5 alkanes of different volatilities within 5 min. Finally, we successfully detected gas mixtures of various polarities using a tandem-column µGC configuration by installing two on-column optical detectors to obtain complementary chromatograms.

Reducing surface area while maintaining implant penetrating profile lowers the brain foreign body response to chronically implanted planar silicon microelectrode arrays.

A consistent feature of the foreign body response (FBR), irrespective of the type of implant, is persistent inflammation at the biotic-abiotic interface signaled by biomarkers of macrophage/microglial activation. Since macrophage-secreted factors shape the foreign body reaction, implant designs that reduce macrophage activation should improve biocompatibility and, with regard to recording devices, should improve reliability and longevity. At present, it is unclear whether the goal of seamless integration is possible or whether electrode developers can modulate specific aspects of the FBR by intentionally manipulating the constitutive properties of the implant. To explore this area, we studied the chronic brain FBR to planar solid silicon microelectrode arrays and planar lattice arrays with identical penetrating profiles but with reduced surface area in rats after an 8-week indwelling period. Using quantitative immunohistochemistry, we found that presenting less surface area after equivalent iatrogenic injury is accompanied by significantly less persistent macrophage activation, decreased blood brain barrier leakiness, and reduced neuronal cell loss. Our findings show that it is possible for implant developers to modulate specific aspects of the FBR by intentionally manipulating the constitutive properties of the implant. Our results also support the theory that the FBR to implanted electrode arrays, and likely other implants, can be explained by the presence of macrophages at the biotic-abiotic interface, which act as a sustained delivery source of bioactive agents that diffuse into the adjacent tissue and shape various features of the brain FBR. Further, our findings suggest that one method to improve the recording consistency and lifetime of implanted microelectrode arrays is to design implants that reduce the amount of macrophage activation at the biotic-abiotic interface and/or enhance the clearance or impact of their released factors.

Evaluation of a microfabricated thermal modulator for comprehensive two-dimensional microscale gas chromatography.

A microfabricated thermal modulator (µTM) designed for ultimate use in a comprehensive two-dimensional microscale gas chromatography (µGC × µGC) system is evaluated. The 2-stage device measures 13 mm (l) × 6 mm (w) × 0.5 mm (h) and consists of two interconnected serpentine etched-Si microchannels suspended from a thin Pyrex cap and wall-coated with PDMS (polydimethylsiloxane). The chip is mounted within a few tens of micrometers of a thermoelectric cooler that maintains both stages at a baseline temperature between -35 and -20 °C in order to focus analytes eluting from an upstream separation column. Each stage is heated to 210 °C sequentially at a rate as high as 2400 °C/s by independent thin-film resistors to inject the analytes in consecutive fractions to a downstream column, and then cooled at a rate as high as -168 °C/s. The average power dissipation is only ∼10 W for heating and 21 W for cooling without using consumable materials. In this study, the outlet of the µTM is connected directly to a flame ionization detector to assess its performance. Following a demonstration of basic operation, the modulated peak amplitude enhancement (PAE) and full-width-at-half-maximum (fwhm) are evaluated for members of a series of n-alkanes (C(6)-C(10)) as a function of the rim and stage temperatures; modulation period, phase, and offset; analyte concentration; and carrier-gas flow rate. A PAE as high as 50 and a fwhm as narrow as 90 ms are achieved for n-octane under optimized conditions.

A 3-d 160-site microelectrode array for cochlear nucleus mapping.

A 3-D application-specific microelectrode array has been developed for physiological studies in guinea pig cochlear nucleus (CN). The batch-fabricated silicon probes contain integrated parylene cables and use a boron etch-stop to define 15µm-thick shanks and limit tissue displacement. Targeting the ventral (three probes) and dorsal (two probes) subnuclei, the custom four-shank 32-site probes are combined in a slotted block platform having a 1.18-mm (2) footprint. The device has permitted, for the first time, high-density 3-D in vivo studies of ventral CN to dorsal CN connections, stimulating with 1000 µm (2) sites in one subnucleus while recording with 177 µm (2) sites in the other. Through these experiments, it has demonstrated the efficacy of bimodal silicon arrays to better understand the central nervous system at the circuit level. The 160 electrode sites also provide a high-density neural interface, which is an essential aspect of auditory prosthesis prototypes.

A dual-shank neural probe integrated with double waveguides on each shank for optogenetic applications.

This paper presents a dual-shank neural probe integrated with double-waveguides on each shank, which enables both optical stimulation and electrical recording. Two 15-µm-thick polymeric (SU-8) waveguides on each neural probe shank have been precisely defined by photolithography with a width of 24 µm and a spacing of 10 µm. The waveguides transmit a light coupled from optical fibers which are placed in the grooves located at the neural probe body. Each shank has 8 iridium recording electrodes which have the area of 11 µm × 13 µm. In front of each waveguide, four recording sites are deployed with a pitch of 100 µm. Blue light (473 nm in wavelength) has been successfully transmitted to the stimulation sites located at the end of the fabricated neural probe tips.

Microfabricated thermal modulator for comprehensive two-dimensional micro gas chromatography: design, thermal modeling, and preliminary testing.

In comprehensive two-dimensional gas chromatography (GC x GC), a modulator is placed at the juncture between two separation columns to focus and re-inject eluting mixture components, thereby enhancing the resolution and the selectivity of analytes. As part of an effort to develop a microGC x microGC prototype, in this report we present the design, fabrication, thermal operation, and initial testing of a two-stage microscale thermal modulator (microTM). The microTM contains two sequential serpentine Pyrex-on-Si microchannels (stages) that cryogenically trap analytes eluting from the first-dimension column and thermally inject them into the second-dimension column in a rapid, programmable manner. For each modulation cycle (typically 5 s for cooling with refrigeration work of 200 J and 100 ms for heating at 10 W), the microTM is kept approximately at -50 degrees C by a solid-state thermoelectric cooling unit placed within a few tens of micrometres of the device, and heated to 250 degrees C at 2800 degrees C s(-1) by integrated resistive microheaters and then cooled back to -50 degrees C at 250 degrees C s(-1). Thermal crosstalk between the two stages is less than 9%. A lumped heat transfer model is used to analyze the device design with respect to the rates of heating and cooling, power dissipation, and inter-stage thermal crosstalk as a function of Pyrex-membrane thickness, air-gap depth, and stage separation distance. Experimental results are in agreement with trends predicted by the model. Preliminary tests using a conventional capillary column interfaced to the microTM demonstrate the capability for enhanced sensitivity and resolution as well as the modulation of a mixture of alkanes.

High-density cochlear implants with position sensing and control.

Silicon-based thin-film technology has been used to develop high-density cochlear electrode arrays with up to 32 sites and four parallel channels of simultaneous stimulation. The lithographically-defined arrays utilize a silicon-dielectric-metal-parylene structure with 180 microm-diameter IrO sites on 250 microm centers. Eight on-board strain gauges allow real-time imaging of array shape during insertion, and a tip sensor measures forces on any structures contacted in the scala tympani (e.g., the basilar membrane). The array can be pre-stressed to hug the modiolus, which provides position reference. Tip position can be resolved to better than 50 microm. Circuitry mounted on the base of the array generates stimulating currents, records intra-cochlear responses and position information, and interfaces with a custom microcontroller and inductively-coupled wireless interface over an eight-lead ribbon cable. The circuitry delivers biphasic 500 microA current pulses with 4 microA resolution and a minimum pulse width of 4 micros. Multiple sites can be driven in parallel to provide higher current levels. Backing structures and articulated insertion tools are being developed for dynamic closed-loop insertion control.

A compact architecture for three-dimensional neural microelectrode arrays.

A new architecture is presented for achieving three-dimensional electronic interfaces to the nervous system using planar microfabricated two-dimensional arrays. This architecture overcomes many of the limitations of existing approaches and enables flexible electrode configurations with minimal overhead in size. A 64-channel (4x4x4) 3-D array using this architecture is demonstrated with sites on 100 microm centers, interfacing with a volume of tissue less than 0.1mm3. The 1mm2 footprint of the device is the smallest ever reported.

A neural amplifier with high programmable gain and tunable bandwidth.

A neural recording amplifier having programmable gain and bandwidth is presented. The gain can be digitally programmed using 6 bits from 100x to 1100x in steps of 100x. The low-frequency cutoff can be varied from less than 10Hz to above 100Hz to accept or reject field potentials while the high-frequency cutoff is fixed at 9kHz. The input referred noise of this amplifier is 4.8microV(rms) and it consumes 50microW operating from +/-1.5V. Implemented in a 0.5microm technology, the amplifier occupies an area of 0.098mm(2). This amplifier has been successfully demonstrated in-vivo and compared to a commercial amplifier.

A fully integrated mixed-signal neural processor for implantable multichannel cortical recording.

A 64-channel neural processor has been developed for use in an implantable neural recording microsystem. In the Scan Mode, the processor is capable of detecting neural spikes by programmable positive, negative, or window thresholding. Spikes are tagged with their associated channel addresses and formed into 18-bit data words that are sent serially to the external host. In the Monitor Mode, two channels can be selected and viewed at high resolution for studies where the entire signal is of interest. The processor runs from a 3-V supply and a 2-MHz clock, with a channel scan rate of 64 kS/s and an output bit rate of 2 Mbps.