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Graphene-based Carbon-Layered Electrode Array Technology for Neural Imaging and Optogenetic Applications

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Graphene-based Carbon-Layered Electrode Array Technology for Neural Imaging and Optogenetic Applications

Dong-Wook Park et al. Nat Commun.

Abstract

Neural micro-electrode arrays that are transparent over a broad wavelength spectrum from ultraviolet to infrared could allow for simultaneous electrophysiology and optical imaging, as well as optogenetic modulation of the underlying brain tissue. The long-term biocompatibility and reliability of neural micro-electrodes also require their mechanical flexibility and compliance with soft tissues. Here we present a graphene-based, carbon-layered electrode array (CLEAR) device, which can be implanted on the brain surface in rodents for high-resolution neurophysiological recording. We characterize optical transparency of the device at >90% transmission over the ultraviolet to infrared spectrum and demonstrate its utility through optical interface experiments that use this broad spectrum transparency. These include optogenetic activation of focal cortical areas directly beneath electrodes, in vivo imaging of the cortical vasculature via fluorescence microscopy and 3D optical coherence tomography. This study demonstrates an array of interfacing abilities of the CLEAR device and its utility for neural applications.

Figures

Figure 1
Figure 1. CLEAR micro-ECoG device.
(a) Basic fabrication process: metal patterning of traces and connection pads on Parylene C/silicon wafer. The silicon wafer is the handling substrate. Transfer and stack four monolayers of graphene sequentially. Graphene patterning to form electrode sites. Second Parylene C deposition and patterning to form device outline. Removal of device from silicon wafer. (b) Diagram of CLEAR device construction showing the layered structures. (c) Demonstration of CLEAR device flexibility. The device is wrapped around a glass bar with a radius of 2.9 mm. (d) Rat-brain-sized CLEAR device: outlined by white dashed line (electrode area of 3.1 × 3.1 mm2). (e) Close-up of rat-sized device showing transparent graphene electrode sites and traces on a Parylene C substrate. This side touches brain surface. Scale bar, 500 μm. (f) Mouse-brain-sized CLEAR device with zero insertion force (ZIF) PCB connector (electrode area of 1.9 × 1.9 mm2).
Figure 2
Figure 2. Electrode characterizations.
(a) Electrical impedance spectra for CLEAR and platinum micro-ECoG devices in saline. The x axis represents real impedance and y axis represents imaginary impedance. Each point was taken at a different frequency, between 10 Hz and 31 kHz. (b) Average CV results over 16 electrode sites on CLEAR, gold and platinum micro-ECoG arrays. (c) Average CV results for 16 electrode sites on CLEAR and gold micro-ECoG arrays. (d) Average artefact effect test results for CLEAR and platinum micro-ECoG devices, with light applied to a single electrode site on each device via an optical fibre attached to a blue laser, with an application of 63.7 mW mm−2 power for 3 ms. (e) Trend of sheet resistance as a function of the number of graphene layer. The error bar represents the s.d. of sheet resistance extracted from five sample measurement. (f) Light transmittance test results for 76 Ω per square four graphene monolayers on a 15-μm Parylene C film (CLEAR), 76 Ω per square four graphene monolayers only (Graphene), Parylene C film only (Parylene), 60 Ω per square ITO/PET and 100 76 Ω per square ITO/PET film. (g) Transmittance versus sheet resistance graph for various conducting materials (graphene, ITO, ultrathin metals).
Figure 3
Figure 3. In vivo-recorded signal characterizations.
(a) Average longitudinal 1 kHz impedance values for CLEAR and platinum micro-ECoG devices implanted in the same animal. The error bar represents s.d. of impedance extracted from 16-channel measurement. (b) Baseline signal power spectra for the CLEAR and platinum devices under dexmedetomidine with 95% confidence interval using jack knife resampling. (c) Baseline signal power spectra for a CLEAR device under two analgesic conditions, dexmedetomidine and isoflurane, compared with an awake condition. (d) Sensory evoked potentials recorded by the CLEAR device via electrical stimulation of the sciatic nerve on the hind leg of the rat, contralateral to the array. The device was implanted over somatosensory cortex. Stimuli were applied for 1 ms at 3 and 1.5 mA current levels. The x scale bar, 50 ms; y scale bar, 100 μV.
Figure 4
Figure 4. Optogenetic experimentation through the transparent CLEAR device.
(a) Schematic drawing of opto-experimental setup, showing the CLEAR device implanted on the cerebral cortex of a mouse, with an optical fibre delivering blue light stimuli to the neural cells. (b) Image of a blue light stimulus being delivered via an optical fibre, through the CLEAR device implanted on the cortex of a Thy1::ChR2 mouse. (c) Optical evoked potentials recorded by the CLEAR device. X-scale bars represent 50 ms, y-scale bars represent 100 μV. (d) Post-mortem control data with the laser set at 24.4 mW mm−2 with the light impingent on electrode site 11 of the CLEAR device. The x scale bar, 50 ms; y scale bars, 100 μV.
Figure 5
Figure 5. Representative in vivo images of the cortical vasculature seen through CLEAR device.
(a) Bright-field image of CLEAR device implanted on the cerebral cortex of a mouse beneath a cranial window. (b) Fluorescence image of same device shown in a. Mouse was given an intravenous injection of fluorescein isothiocyanate–dextran to fluorescently label the vasculature. (c,d) Higher magnification bright-field and fluorescence images of same device shown in a and b, respectively. (e,f) Bright-field and fluorescence images of standard rat-sized micro-ECoG arrays with platinum electrode sites, respectively. Scale bars, 500 μm (a,b), 250 μm (c,d), 750 μm (e,f). In vivo vasculature imaging was repeated in three rats, each with a CLEAR and platinum microECoG array. Images were representative of presented and previously published data.
Figure 6
Figure 6. Optical coherence tomography through CLEAR and platinum devices.
(a,c) Maximum intensity projection (MIP) of OCT angiogram showing cortical vasculature visible through the CLEAR micro-ECoG device (FOV 2.8 × 2.8 mm2 and 1.1 × 1.1 mm2, respectively). (b,d) Doppler blood flow velocity image showing the directionality of blood flowing through the vasculature below the CLEAR device (FOV 2.8 × 2.8 mm2 and 1.1 × 1.1 mm2). Red colour represents blood flowing towards the lens and green colour represents blood flowing away. (e) Cross-sectional angiogram after contrast enhancement and de-shadowing (CLEAR device). (f) 3D visualization of the vasculature (red colour) overlaid on the structural data (grey) (CLEAR device). (g) MIP of angiogram through a platinum micro-ECoG device. (h) Corresponding Doppler blood flow velocity measurements. (i) Corresponding cross-sectional angiogram. (j) Corresponding 3D visualization of vessel structure for a micro-ECoG array with platinum electrode sites. Scale bars, 200 μm (a,b), 100 μm (ci).

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