Histological assessment of a chronically implanted cylindrically-shaped, polymer-based neural probe in the monkey.

Objective.Previous studies demonstrated the possibility to fabricate stereo-electroencephalography probes with high channel count and great design freedom, which incorporate macro-electrodes as well as micro-electrodes offering potential benefits for the pre-surgical evaluation of drug resistant epileptic patients. These new polyimide probes allowed to record local field potentials, multi- and single-unit activity (SUA) in the macaque monkey as early as 1 h after implantation, and yielded stable SUA for up to 26 d after implantation. The findings opened new perspectives for investigating mechanisms underlying focal epilepsy and its treatment, but before moving to possible human application, safety data are needed. In the present study we evaluate the tissue response of this new neural interface by assessing post-mortem the reaction of brain tissue along and around the probe implantation site.Approach.Three probes were implanted, independently, in the brain of one monkey (Macaca mulatta) at different times. We used specific immunostaining methods for visualizing neuronal cells and astrocytes, for measuring the extent of damage caused by the probe and for relating it with the implantation time.Main results.The size of the region where neurons cannot be detected did not exceed the size of the probe, indicating that a complete loss of neuronal cells is only present where the probe was physically positioned in the brain. Furthermore, around the probe shank, we observed a slightly reduced number of neurons within a radius of 50µm and a modest increase in the number of astrocytes within 100µm.Significance.In the light of previous electrophysiological findings, the present data suggest the potential usefulness and safety of this probe for human applications.

Cardiac pacing using transmural multi-LED probes in channelrhodopsin-expressing mouse hearts.

Optogenetics enables cell-type specific monitoring and actuation via light-activated proteins. In cardiac research, expressing light-activated depolarising ion channels in cardiomyocytes allows optical pacing and defibrillation. Previous studies largely relied on epicardial illumination. Light penetration through the myocardium is however problematic when moving to larger animals and humans. To overcome this limitation, we assessed the utility of an implantable multi light-emitting diode (LED) optical probe (IMLOP) for intramural pacing of mouse hearts expressing cardiac-specific channelrhodopsin-2 (ChR2). Here we demonstrated that IMLOP insertion needs approximately 20 mN of force, limiting possible damage from excessive loads applied during implantation. Histological sections confirmed the confined nature of tissue damage during acute use. The temperature change of the surrounding tissue was below 1 K during LED operation, rendering the probe safe for use in situ. This was confirmed in control experiments where no effect on cardiac action potential conduction was observed even when using stimulation parameters twenty-fold greater than required for pacing. In situ experiments on ChR2-expressing mouse hearts demonstrated that optical stimulation is possible with light intensities as low as 700 µW/mm2; although stable pacing requires higher intensities. When pacing with a single LED, rheobase and chronaxie values were 13.3 mW/mm2 ± 0.9 mW/mm2 and 3 ms ± 0.6 ms, respectively. When doubling the stimulated volume the rheobase decreased significantly (6.5 mW/mm2 ± 0.9 mW/mm2). We have demonstrated IMLOP-based intramural optical pacing of the heart. Probes cause locally constrained tissue damage in the acute setting and require low light intensities for pacing. Further development is necessary to assess effects of chronic implantation.

Smart bracket for multi-dimensional force and moment measurement.

Atraumatic, well-directed, and efficient tooth movement is interrelated with the therapeutic application of adequately dimensioned forces and moments in all three dimensions. The lack of appropriate monitoring tools inspired the development of an orthodontic bracket with an integrated microelectronic chip equipped with multiple piezoresistive stress sensors. Such a 'smart bracket' was constructed (scale of 2.5:1) and calibrated. To evaluate how accurately the integrated sensor system allowed for the quantitative determination of three-dimensional force-moment systems externally applied to the bracket, we exerted 396 different force-moment combinations with dimensions within usual therapeutic ranges (+/- 1.5 N and +/- 15 Nmm). Comparison between the externally applied force-moment components and those reconstructed on the basis of the stress sensor signals revealed very good agreement, with standard deviations in the differences of 0.037 N and 0.985 Nmm, respectively. We conclude that our methodological approach is generally suitable for monitoring the relatively low forces and moments exerted on individual teeth with fixed orthodontic appliances.

Versatile, modular 3D microelectrode arrays for neuronal ensemble recordings: from design to fabrication, assembly, and functional validation in non-human primates.

OBJECTIVE: Application-specific designs of electrode arrays offer an improved effectiveness for providing access to targeted brain regions in neuroscientific research and brain machine interfaces. The simultaneous and stable recording of neuronal ensembles is the main goal in the design of advanced neural interfaces. Here, we describe the development and assembly of highly customizable 3D microelectrode arrays and demonstrate their recording performance in chronic applications in non-human primates. APPROACH: System assembly relies on a microfabricated stacking component that is combined with Michigan-style silicon-based electrode arrays interfacing highly flexible polyimide cables. Based on the novel stacking component, the lead time for implementing prototypes with altered electrode pitches is minimal. Once the fabrication and assembly accuracy of the stacked probes have been characterized, their recording performance is assessed during in vivo chronic experiments in awake rhesus macaques (Macaca mulatta) trained to execute reaching-grasping motor tasks. MAIN RESULTS: Using a single set of fabrication tools, we implemented three variants of the stacking component for electrode distances of 250, 300 and 350 µm in the stacking direction. We assembled neural probes with up to 96 channels and an electrode density of 98 electrodes mm-2. Furthermore, we demonstrate that the shank alignment is accurate to a few µm at an angular alignment better than 1°. Three 64-channel probes were chronically implanted in two monkeys providing single-unit activity on more than 60% of all channels and excellent recording stability. Histological tissue sections, obtained 52 d after implantation from one of the monkeys, showed minimal tissue damage, in accordance with the high quality and stability of the recorded neural activity. SIGNIFICANCE: The versatility of our fabrication and assembly approach should significantly support the development of ideal interface geometries for a broad spectrum of applications. With the demonstrated performance, these probes are suitable for both semi-chronic and chronic applications.

Chronic neural probe for simultaneous recording of single-unit, multi-unit, and local field potential activity from multiple brain sites.

OBJECTIVE: Drug resistant focal epilepsy can be treated by resecting the epileptic focus requiring a precise focus localisation using stereoelectroencephalography (SEEG) probes. As commercial SEEG probes offer only a limited spatial resolution, probes of higher channel count and design freedom enabling the incorporation of macro and microelectrodes would help increasing spatial resolution and thus open new perspectives for investigating mechanisms underlying focal epilepsy and its treatment. This work describes a new fabrication process for SEEG probes with materials and dimensions similar to clinical probes enabling recording single neuron activity at high spatial resolution. APPROACH: Polyimide is used as a biocompatible flexible substrate into which platinum electrodes and leads are integrated with a minimal feature size of 5 µm. The polyimide foils are rolled into the cylindrical probe shape at a diameter of 0.8 mm. The resulting probe features match those of clinically approved devices. Tests in saline solution confirmed the probe stability and functionality. Probes were implanted into the brain of one monkey (Macaca mulatta), trained to perform different motor tasks. Suitable configurations including up to 128 electrode sites allow the recording of task-related neuronal signals. MAIN RESULTS: Probes with 32 and 64 electrode sites were implanted in the posterior parietal cortex. Local field potentials and multi-unit activity were recorded as early as one hour after implantation. Stable single-unit activity was achieved for up to 26 days after implantation of a 64-channel probe. All recorded signals showed modulation during task execution. SIGNIFICANCE: With the novel probes it is possible to record stable biologically relevant data over a time span exceeding the usual time needed for epileptic focus localisation in human patients. This is the first time that single units are recorded along cylindrical polyimide probes chronically implanted 22 mm deep into the brain of a monkey, which suggests the potential usefulness of this probe for human applications.

Microsystems in medicine.

The complexity of modern surgical and analytical methods requires the miniaturisation of many medical devices. The LIGA technique and also mechanical microengineering are well known for the batch fabrication of microsystems. Actuators and sensors are developed based on these techniques. The hydraulic actuation principle is advantageous for medical applications since the energy may be supplied by pressurised balanced salt solution. Some examples are turbines, pumps and valves. In addition, optical sensors and components are useful for analysis and inspection as represented by microspectrometers and spherical lenses. Finally, plastic containers with microporous bottoms allow a 3-dimensional growth of cell culture systems.

Modular assembly concept for 3D neural probe prototypes offering high freedom of design and alignment precision.

The new assembly technology developed in this research provides a means to extend planar intracortical neural probes with one-dimensional (1D) and two-dimensional (2D) electrode arrangements into complex three-dimensional (3D) neural probes. The approach is based on novel silicon stacking modules realized using microsystems technologies. With these microcomponents, 3D probes can be assembled flexibly and tailored to the demands of neuroscientific experiments. The manufacturing process of the stacking modules provides the possibility to adjust the electrode spacing in the stacking direction with micrometer precision. The assembly method is demonstrated with 32-channel systems comprising 7-mm-long and 50-µm-thin neural probes. The angular alignment between the neural probes and their stacking modules after assembly as well as the vertical electrode pitch were determined to be about 1° and 353±15 µm, respectively.

Miniaturized tool for optogenetics based on an LED and an optical fiber interfaced by a silicon housing.

This paper reports on the design, simulation, fabrication and characterization of a tool for optogenetic experiments based on a light emitting diode (LED). A minimized silicon (Si) interface houses the LED and aligns it to an optical fiber. With a Si housing size of 550×500×380 µm(3) and an electrical interconnection of the LED by a highly flexible polyimide (PI) ribbon cable is the system very variable. PI cables and Si housings are fabricated using established microsystem technologies. A 270×220×50 µm(3) bare LED chip is flip-chip-bonded onto the PI cable. The Si housing is adhesively attached to the PI cable, thereby hosting the LED in a recess. An opposite recess guides the optical fiber with a diameter of 125 µm. An aperture in-between restricts the emitted LED light to the fiber core. The optical fiber is adhesively fixed into the Si housing recess. An optical output intensity at the fiber end facet of 1.71 mW/mm(2) was achieved at a duty cycle of 10 % and a driving current of 30 mA.

Fabrication and characterization of a high-resolution neural probe for stereoelectroencephalography and single neuron recording.

This paper reports on the design, fabrication, and characterization of neural probes for stereoelectroencephalography (SEEG). The probe specifically targets focal epilepsy as key application. However, probes of this type can also be used for the diagnosis and treatment of other neural dysfunctions such as Parkinson's disease or tremor, typically requiring deep brain probes. The probe fabrication, of which most processes are parallel batch processes, relies on a novel fabrication concept for rolling and gluing thin film polyimide sheets with integrated electrodes into permanent cylindrical shapes with diameters down to 800 µm. The SEEG probes, comprise several macro-electrodes designed to record local field potentials, and micro-electrodes positioned in-between, dedicated to monitoring single unit activity, with a total channel count of 32, despite the small diameter. While platinum micro-electrodes with a diameter of 35 µm have impedances of about 255 kΩ at 1 kHz, impedance values down to about 1.5 kΩ have been measured for the macro-electrodes. The devices have shown good compatibility with magnetic resonance imaging in a 9.4 T magnet, enabling the precise post-operative probe localization within the brain.

FIB preparation and SEM investigations for three-dimensional analysis of cell cultures on microneedle arrays.

We report the investigation of the interfaces between microneedle arrays and cell cultures in patch-on-chip systems by using Focused Ion Beam (FIB) preparation and Scanning Electron Microscopy (SEM). First, FIB preparations of micro chips are made to determine the size and shape of the designed microneedles. In this essay, we investigate the cell-substrate interaction, especially the cell adhesion, and the microneedle's potential cell penetration. For this purpose, cross-sectional preparation of these hard/soft hybrid structures is performed by the FIB technology. By applying the FIB technology followed by high-resolution imaging with SEM, new insights into the cell-substrate interface can be received. One can clearly distinguish between cells that are only in contact with microneedles and cells that are penetrated by microneedles. A stack of slice images is collected by the application of the slice-and-view setup during FIB preparation and is used for three-dimensional reconstruction of cells and micro-needles.

Torsional bridge setup for the characterization of integrated circuits and microsensors under mechanical shear stress.

We present a torsional bridge setup for the electro-mechanical characterization of devices integrated in the surface of silicon beams under mechanical in-plane shear stress. It is based on the application of a torsional moment to the longitudinal axis of the silicon beams, which results in a homogeneous in-plane shear stress in the beam surface. The safely applicable shear stresses span the range of ±50 MPa. Thanks to a specially designed clamping mechanism, the unintended normal stress typically stays below 2.5% of the applied shear stress. An analytical model is presented to compute the induced shear stress. Numerical computations verify the analytical results and show that the homogeneity of the shear stress is very high on the beam surface in the region of interest. Measurements with piezoresistive microsensors fabricated using a complementary metal-oxide-semiconductor process show an excellent agreement with both the computational results and comparative measurements performed on a four-point bending bridge. The electrical connection to the silicon beam is performed with standard bond wires. This ensures that minimal forces are applied to the beam by the electrical interconnection to the external instrumentation and that devices with arbitrary bond pad layout can be inserted into the setup.

Biosensor microprobes with integrated microfluidic channels for bi-directional neurochemical interaction.

This paper reports on silicon-based microprobes, 8 mm long and 250 µm × 250 µm cross-section, comprising four recessed biosensor microelectrodes (50 µm × 150 µm) per probe shank coated with an enzymatic layer for the selective detection of choline at multiple sites in brain tissue. Integrated in the same probe shank are up to two microfluidic channels for controlled local liquid delivery at a defined distance from the biosensor microelectrodes. State-of-the-art silicon micromachining processing was applied for reproducible fabrication of these experiment-tailored multi-functional probe arrays. Reliable electric and fluidic interconnections to the microprobes are guaranteed by a custom-made holder. The reversible packaging method implemented in this holder significantly reduces cost and assembly time and simplifies storage of the biosensor probes between consecutive experiments. The functionalization of the electrodes is carried out using electrochemically aided adsorption. This spatially controlled deposition technique enables a parallel deposition of membranes and is especially useful when working with microelectrode arrays. The achieved biosensors show adequate characteristics to detect choline in physiologically relevant concentrations at sufficient temporal and spatial resolution for brain research. Sensitivity to choline better than 10 pA µm(-1), detection limit below 1 µM and response time of 2 s were obtained. The proposed combination of biosensors and microfluidic injectors on the same microprobe allows simultaneous chemical stimulation and recording as demonstrated in an agarose gel-based brain phantom.

Two-dimensional multi-channel neural probes with electronic depth control.

This paper presents multi-electrode arrays for in vivo neural recording applications incorporating the principle of electronic depth control (EDC), i.e., the electronic selection of recording sites along slender probe shafts independently for multiple channels. Two-dimensional (2D) arrays were realized using a commercial 0.5- µm complementary-metal-oxide-semiconductor (CMOS) process for the EDC circuits combined with post-CMOS micromachining to pattern the comb-like probes and the corresponding electrode metallization. A dedicated CMOS integrated front-end circuit was developed for pre-amplification and multiplexing of the neural signals recorded using these probes.

Discrete cortical responses from multi-site supra-choroidal electrical stimulation in the feline retina.

Exploration into electrical stimulation of the retina has thus far focussed primarily upon the development of prostheses targeted at one of two sites of intervention - the epi- and sub-retinal surfaces. These two approaches have sound, logical merit owing to their proximity to retinal neurons and their potential to deliver stimuli via the surviving retinal neural networks respectively. There is increasing evidence, however, that electric field effects, electrode engineering limitations, and electrode-tissue interactions limit the spatial resolution that once was hoped could be elicited from electrical stimulation at epi- and sub-retinal sites. An alternative approach has been proposed that places a stimulating electrode array within the supra-choroidal space - that is, between the sclera and the choroid. Here we investigate whether discrete, cortical activity patterns can be elicited via electrical stimulation of a feline retina using a custom, 14 channel, silicone rubber and Pt electrode array arranged in two hexagons comprising seven electrodes each. Cortical responses from Areas 17/18 were acquired using a silicon-based, multi-channel, penetrating probe developed at IMTEK, University of Freiburg, within the European research project NeuroProbes. Multi-unit spike activity was recorded in synchrony with the presentation of electrical stimuli. Results show that distinct cortical response patterns could be elicited from each hexagon separated by 1.8 mm (center-to-center) with a center-to-center electrode spacing within each hexagon of 0.55 mm. This lends support that higher spatial resolution may also be discerned.

Hybrid microprobes for chronic implantation in the cerebral cortex.

This paper reports on a neural device for chronic implantation into the cerebral cortex. Silicon microprobes with 36 electrodes arranged on four shafts are fabricated using MEMS technology. The hybrid integration of a ribbon cable with high flexibility provides the connection of the electrodes to external instrumentation. Crosstalk between the channels is investigated, as well as the electrode stability for a time period of one month in vitro. Due to the geometry and the mechanical stability of these microprobes, insertions are possible without the need for prior opening of the dura mater. A dedicated insertion tool has been fabricated to achieve a precise insertion of the microprobes and their subsequent mechanical decoupling. Additionally, a protection chamber allowing the secure attachment of two connector units on the skull is introduced. The short-time chronic implantation of microprobes showed that neural activity can be recorded, including single unit activity, which was present after four days.

A 3D slim-base probe array for in vivo recorded neuron activity.

This paper introduces the first experimental results of a new implantable slim-base three-dimensional (3D) probe array for cerebral applications. The probes are assembled perpendicularly into the slim-base readout platform where electrical and mechanical connections are achieved simultaneously. A new type of micromachined interconnect has been developed to establish electrical connection using extreme planarization techniques. Due to the modular approach of the platform, probe arrays of different dimensions and functionality can be assembled. The platform is only several hundred microns thick which is highly relevant for chronic experiments in which the probe array should be able to float on top of the brain. Preliminary tests were carried out with the implantation of a probe array into the auditory cortex of a rat.

Microprobe array with low impedance electrodes and highly flexible polyimide cables for acute neural recording.

This paper reports on a novel type of silicon-based microprobes with linear, two and three dimensional (3D) distribution of their recording sites. The microprobes comprise either single shafts, combs with multiple shafts or 3D arrays combining two combs with 9, 36 or 72 recording sites, respectively. The electrical interconnection of the probes is achieved through highly flexible polyimide ribbon cables attached using the MicroFlex Technology which allows a connection part of small lateral dimensions. For an improved handling, probes can be secured by a protecting canula. Low-impedance electrodes are achieved by the deposition of platinum black. First in vivo experiments proved the capability to record single action potentials in the motor cortex from electrodes close to the tip as well as body electrodes along the shaft.

B.CAT CONFIRM, a rapid test for confirmation of Branhamella catarrhalis.

B.CAT CONFIRM (Scott Laboratories, Inc., Fiskeville, R.I.), a rapid test for detection of tributyrin hydrolysis, was evaluated for its ability to identify strains of Branhamella catarrhalis and to differentiate them from Neisseria species and related species. On initial testing, B.CAT CONFIRM was positive for 65 (96%) of 68 B. catarrhalis strains within 30 min after inoculation. Retesting of the remaining three strains resulted in their correct identification. B.CAT CONFIRM was negative for all Neisseria spp. (130 strains) and for Kingella spp. (3 strains). Two of the three Moraxella spp. were weakly positive in the B.CAT CONFIRM after 60 min, but these reactions were easily distinguishable from the strong reactions of B. catarrhalis strains. This test will be helpful in the clinical laboratory for the rapid identification of B. catarrhalis in clinical specimens.

Identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria with the MicroScan Haemophilus-Neisseria identification panel.

The Haemophilus-Neisseria identification (HNID) panel (American MicroScan, Sacramento, Calif.) is a 4-h microdilution format system for identification of Haemophilus and Neisseria spp., Branhamella (Moraxella) catarrhalis, and Gardnerella vaginalis. The HNID panel was evaluated by using 423 clinical isolates and stock strains of these organisms, and HNID identifications were compared with those obtained by conventional methods. In addition, 32 isolates representing six genera not included in the HNID data base were tested to determine whether these organisms would produce unique biotype numbers for possible inclusion in the data base. The HNID panel correctly identified 95.3% of 86 Neisseria gonorrhoeae strains, 96% of 25 G. vaginalis strains, and 100% of 28 Neisseria lactamica strains and 48 B. catarrhalis strains. Only 64.7% of 68 Neisseria meningitidis isolates were identified correctly owing to false-negative or equivocal carbohydrate and/or aminopeptidase reactions. Among the Haemophilus spp., 98.8% of 83 H. influenzae strains, 97.1% of 34 H. parainfluenzae strains, and 80% of 15 H. aphrophilus and H. paraphrophilus strains were correctly identified. Eight strains of Neisseria cinerea, a species not included in the data base, produced profiles identical with those for B. catarrhalis and N. gonorrhoeae. Isolates of other species not included in the data base, including Eikenella corrodens, Kingella spp., and Cardiobacterium hominis, produced unique biochemical reaction patterns on the panel. Modification of interpretative criteria for certain tests, expansion of the data base to include other species, and suggestions for additional confirmatory tests will increase the accuracy and utility of the HNID panel.

Comparison of monoclonal antibody methods and a ribosomal ribonucleic acid probe test for Neisseria gonorrhoeae culture confirmation.

Recently, a chemiluminescent nucleic acid probe test that specifically detects the ribosomal ribonucleic acid of Neisseria gonorrhoeae has been released for clinical laboratory use (AccuProbe Neisseria gonorrhoeae). In this study, three coagglutination tests (GonoGen I, Meritec GC, and GC Omni), the GonoGen II immunofiltration method and the Micro Trak Neisseria gonorrhoeae fluorescent monoclonal antibody test were compared with AccuProbe for identification of gonococci. Strains tested (n = 376) included 194 Neisseria gonorrhoeae, 82 Neisseria meningitidis, 32 Neisseria lactamica, 32 Neisseria species, 32 Moraxella catarrhalis, 2 Moraxella spp. and 2 Kingella denitrificans. The GonoGen I, Meritec GC and GC Omni coagglutination tests produced clearly positive results for 93.8%, 92.3% and 95.9% of the gonococci, respectively. The GonoGen II unequivocally identified 91.8% and the MicroTrak fluorescent antibody test identified 90.7% with 2+ or greater fluorescence. AccuProbe identified 100% of the gonococci tested. GonoGen I and GonoGen II were 98% specific, Meritec GC was 99% specific and the specificity of the GC Omni, MicroTrak fluorescent antibody and AccuProbe tests was 100%. While antibody-based tests were reliable when results were clearly interpretable, the AccuProbe was the only confirmatory test that was 100% accurate. Serotyping studies indicate that an array of beta-lactamase positive and negative gonococcal serotypes fail to react with the monoclonal antibody-based tests in general and with the fluorescent antibody test in particular.