CLEAR device fabrication

Following graphene characterizations using Raman spectroscopy (as described in the Supplementary Information), implantable graphene neural electrode arrays were fabricated on a 4-inch silicon wafer. Figure 1a shows a simplified schematic of the fabrication process. Briefly, wafers were coated with Parylene C films using a chemical vapour deposition process. Next, the connection pads and initial portions of the traces were patterned with gold via electron beam evaporation and lift-off techniques. The use of gold for the traces and pads was necessary to ensure a good mechanical connection to the zero insertion force printed circuit board (PCB) connectors used for reading the brain signals into the computer. The electrode sites and portions of the traces that were to be in contact with the brain were left for the subsequent graphene transfer and patterning, such that the brain contact area of the electrode would be transparent. Following metallization, four graphene monolayers were transferred and stacked sequentially onto the wafer surface, using the wet transfer technique described in Supplementary Figs S1–S4. A sacrificial layer of SiO 2 was then deposited to protect the graphene layers from being etched in the reactive ion etching (RIE) steps to follow. After that, the graphene was patterned to form the electrode sites using RIE and another insulating layer of Parylene C was deposited. RIE was then employed to expose the electrode sites and pads, and form the array outlines. Finally, the devices were peeled from the wafer, the SiO 2 protection layer was removed by wet etching and the arrays were inserted into the PCB connectors. The detailed fabrication process and the graphene characterization using Raman spectroscopy are described in Supplementary Figs S5 and S6, respectively.

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). Full size image

Device characterization

The patency of the completed devices was verified via electrical impedance spectroscopy. Impedance spectra were obtained for each electrode site using an Autolab PGSTAT12 potentiostat. Devices were connected to the Autolab machine via the PCB connectors, which were connected to a passive 32-channel Tucker-Davis Technologies (TDT) head stage. Impedances were evaluated at 30 different frequencies, ranging from 10 to 30,937 Hz. If electrode sites had impedance values <600 kΩ at 1 kHz frequency, they were considered to be viable for implantation. One kilohertz frequency was selected for evaluation, because it is known to be a common benchmark for neural impedance analysis22,23. Figure 2a shows representative electrical impedance spectra for CLEAR and traditional platinum micro-ECoG devices tested in saline. It is apparent from the plot that the phase angle is higher in the case of the CLEAR device. This means that the value of the reactance is higher for the graphene sites than for the platinum. However, the average magnitude of the impedance at 1 kHz frequency was only slightly higher for the CLEAR device than for the platinum array (243.5±5.9 kΩ for graphene versus 188.8±92.9 kΩ for platinum). As the signal-recording apparatus involves the use of a high-input impedance amplifier, the reactance difference is not expected to have adverse effects on the signal recordings.

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). Full size image

In addition to impedance spectroscopy, cyclic voltammetry (CV) was performed on select CLEAR devices and on devices with gold and platinum electrode sites for comparison purposes. CV was also performed using the Autolab system. CV scans were taken from −0.6 to 0.8 V with a step potential of 0.0105 V and a scan rate of 0.0500 V s−1 (refs 24, 25). The voltage range was chosen so as to stay within the water window. Average CV curves for gold, platinum and CLEAR devices are shown in Fig. 2b,c. The CV results for the platinum device were drastically different than those for either the gold or the CLEAR devices. Although this demonstrates that the platinum electrode sites have a greater charge-carrying capacity, it does not rule out the use of CLEAR devices for neural interfacing applications. In fact, the average CV curves for the CLEAR and gold electrode sites were similar. As gold has proven itself as a reliable material for recording electrode sites26,27, this suggests that the graphene conductors will also be sufficient.

The construction of completely transparent neural surface electrode arrays enables unique research capabilities in combining optogenetics28 with electrophysiology, by allowing for delivery of light stimuli through the array, directly to the brain region from which the recordings are obtained. Even though the electrodes are optically transparent, an artefact is still present when high-intensity light is applied directly onto an electrode site. The artefact is generally smaller in amplitude and temporally different than the optogenetically evoked neural signal. The artefact is reproducible and can be characterized by testing the electrode site in saline, implanting into wild-type animals or testing in cadaver experiments. To test the artefact, the devices were placed face down in saline solution and a 200-μm optical fibre connected to a 100-mW, 473-nm diode laser was used to shine light onto the backs of the electrode sites. The light pulses were delivered by applying 3 V to the laser for 3 ms (up to 63.7 mW mm−2). Figure 2d shows the electrical pulses elicited by the light impingent on graphene and platinum sites. From the plot, it appears that the amplitude of the artefact is similar for both the graphene and the platinum sites, although the platinum electrode returns to baseline more quickly. We expect the main mechanism of the artefact is similar to when light is applied to a conventional metal electrode, which is known as Becquerel effect, a classical photoelectrochemical effect first demonstrated by Becquerel in 1839 (refs 29, 30). More details are discussed in the optogenetic testing section with further in vivo experiments and analysis.

Another important characteristic for optogenetic application of the CLEAR device is the amount of light transmitted through the graphene electrode sites and Parylene substrate. As the intention for the transparent device is that one can both image and project light onto the underlying brain tissue, it is important that a large percentage of the light impinging on the array is transmitted through the device. In a material, inherent tradeoffs exist between transparency and conductivity. In general, by increasing the thickness of a material, the conductivity is increased but the transparency is decreased. This tradeoff also exists in the graphene case. Bae et al.21 reported an increase in conductance (that is, sheet resistance decrease) and a decrease in transmittance when going from one to four graphene monolayers. To verify this trend in our chemical vapor deposition (CVD) graphene, sheet resistance was extracted using the transmission line method (Supplementary Fig. S7). Figure 2e shows the sheet resistance decrease from 152 to 76 Ω per square for one-layer and four-layer graphene. This shows a similar sheet resistance trend to the previous report. Note that relatively higher values than the reported values are attributed to their HNO 3 -doped graphene21. For the CLEAR device, four-layer graphene was chosen to have the minimum sheet resistance while maintaining around 90% transmittance. In addition, the four-layer graphene is expected to have better yield than fewer-layer graphene by compensating possible mechanical tear-off of each graphene layer during the transfer process.

A plot of the light transmittance versus wavelength for Parylene, four-layer graphene, CLEAR (four-layer graphene/parylene) and ITO/PET (polyethylene terephthalate) films is shown in Fig. 2f. The measurements were taken for light wavelengths from 300 to 1,500 nm using a ultraviolet–visible spectrometer. The sinusoidal shape of the transmittance curve is common for the Parylene C material31. For the CLEAR device, an average of ~90% of the light impinging on the substrate is transmitted at the desired wavelengths (470 nm for excitation of channelrhodopsin and 570 nm for halorhodopsin). This is similar to previously reported results and is sufficient for numerous optogenetic and imaging applications9,32. To compare the CLEAR and ITO films, commercial ITO/PET films with similar sheet resistance values (60 and 100 Ω per square) were measured using the same instrument. For both films, an average of around 80% transmittance in the visible-infrared range and dramatically reduced transmittance near the ultraviolet range are shown. In addition, a transmittance difference of ~10% is shown in the 420–440 nm range, which is attributed to the different properties of the ITO films.

For a more quantitative comparison between the graphene, ITO and ultrathin metal films, the measured transmittance and sheet resistance values are plotted in Fig. 2g, along with various values reported in the literature. Sheet resistance is widely used to evaluate and compare conducting materials13,14,21,33,34. From the plot we can see that for the given sheet resistance range, graphene films show >90% transmittance while ITO and ITO/PET films show closer to 80% transmittance. Moreover, the ultra-thin metals show much less transmittance (~60%). This comparison indicates that the light transmittance through the CLEAR devices is superior to ITO and ultra-thin metals.

In vivo validation of neural signal and impedance recordings

To demonstrate the in vivo performance of the CLEAR devices, the arrays were implanted in four rats and five mice, one wild-type mouse for imaging and four Thy1::ChR2 mice for imaging and optogenetic testing. Table 1 describes the type of implantation performed in each case and the type of data collected from each animal. Three of the rat implantations were bilateral, with CLEAR and the platinum devices implanted within the same animal for a fair comparison and to reduce overall animal numbers.

Table 1 Number of in vivo experiments for each purpose Full size table

Average electrode site impedance (measured from the first day of device implantation) for CLEAR and platinum micro-ECoG arrays implanted in the same animal are plotted over time in Fig. 3a. Both devices experienced a steep rise in impedance within the first 10 days after implantation, most probably due to the initial tissue response to the implanted arrays22. After this initial sharp increase, the impedances appear to have plateaued, with some minor day-to-day fluctuations. The shape of these impedance curves is characteristic of epidurally implanted micro-ECoG devices7. There was no statistically significant difference between the impedance changes for the CLEAR and platinum micro-ECoG devices over the entire implantation period, suggesting that the CLEAR device will perform, as well as the platinum device over the explored time period.

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. Full size image

Figure 3b shows power spectra for the baseline local field potentials recorded by single channels on the CLEAR and platinum micro-ECoG devices in the 1–100 Hz frequency range. As with the longitudinal impedance data, there is little difference between the signals recorded by the two different arrays. The 95% confidence intervals for the CLEAR and platinum devices overlap, suggesting little difference between the signals recorded by the different devices. This similarity is shown in the high frequency range (1–600 Hz) as well (Supplementary Fig. S8a). In addition, Fig. 3c shows the baseline spectra obtained with the animals anaesthetized using two different anaesthetics, dexmedetomidine and isoflurane. Dexmedetomidine induces sleep-like rhythms, with more low frequency oscillations, while isoflurane suppresses general signal power, as shown in the figure. Both dexmedetomidine and isoflurane suppress high gamma −6 dB at 80 Hz, a 75% reduction in amplitude compared with the awake condition. This suggests that clear electrodes are able to pick up the high-gamma signals that have been found to be useful for ECoG-based brain–computer interface applications35,36.

In addition to baseline signal recordings and impedance spectroscopy measurements, rats were tested for electrical-evoked potentials. In these experiments, the hindlimbs of the animals were stimulated with surface electrodes placed above and below the sciatic nerve. Stimuli consisted of 1 ms biphasic electrical pulses, with amplitudes varying from 1 to 3 mA. Evoked potentials were recorded with stimuli applied both ipsilateral and contralateral to the implanted devices, to verify that the result was, in fact, the somatosensory response to the electrical stimulus. If this was true, evoked potentials would be seen only when the stimuli were applied contralateral to the implanted electrode array, due to the crossing of the neural pathways in the brainstem and spinal cord. Figure 3d shows a summary of the evoked potential results for the contralateral implanted CLEAR device at two stimulation levels. The results show a neural response with ~100 μV evoked potentials. Signals recorded in the device with ipsilateral leg stimulation lacked the neural evoked potential that was found on the contralateral side (Supplementary Fig. S8b). In general, the results displayed in Fig. 3 show that the graphene electrode sites are capable of recording both spontaneous baseline activity and evoked neural signals with the same level of clarity as the platinum sites, and generally similar impedance behaviour and stability over time.

Optogenetic testing

Three Thy1::ChR2 mice were terminally implanted with a CLEAR device for the purpose of optogenetic evaluation. These mice had neurons expressing the channelrhodopsin-2 protein, making them susceptible to excitation when exposed to blue (473 nm) light. The average evoked response is shown for three different light-intensity stimulation levels in Fig. 4c. The stimulus time was 3 ms. The initial peak corresponds to the expected stimulus artefact and the second, longer peak is the evoked neural response. The stimulus artefact peak is detected ~3 ms after the stimulus onset, while the evoked neural response peak has latency ~7.7 ms following the stimulus onset. These latencies are highly dependent on the details of the eventual intended experimental requirements (that is, light intensity, stimulus duration and interstimulus interval). This leads to the logical consequence that in many experimental paradigms the stimulus artefact will be completely distinguishable from the evoked neural response, and in others there may be considerable overlap.

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. Full size image

Once experimentation was complete, the animals were killed and a control experiment was conducted with the CLEAR micro-ECoG array placed on the somatosensory cortex of the killed animal to verify that the signals recorded were from neurons affected by the light stimulation and not due to the artefact. From Fig. 4d we can see that the signals obtained from the post-mortem control experiment are different from the signals recorded from the living animal, both temporally and according to amplitude of the negative peaks. These results demonstrate that the signals in Fig. 4c were evoked neural responses to the light stimulation.

An experiment to demonstrate the increased spatial resolution of the CLEAR device was performed using a lower-power light intensity of 1.24 mW mm−2. As this lower-power light results in less spatial spread, the volume of neural tissue activated is also more confined and its response could be detected in a more focal area. Supplementary Fig. S9a shows that with low-intensity light stimulation it is possible to activate a relatively focal brain region (predominantly on one electrode site in this case). In this case, the amplitude of neural response was also reduced compared with the higher light intensity used in Supplementary Fig. S9b (24.4 mW mm−2). This is attributed to the reduced volume of stimulated neurons caused by the reduced radius and depth of the light stimulation37.

For more detailed artefact analysis, we investigated the dependency of the light stimulation timing and power. Optical stimulus artefact is an important consideration for any kind of recording electrode and is difficult to avoid due to the ionic charge transfer layer at the conductor–electrolyte interface. Electrically conductive materials in ionic solution are in general subject to the Becquerel effect, a classical photoelectrochemical effect first demonstrated by Becquerel in 1839 and known as the main mechanism of the stimulus artefact for conventional metal electrodes29,30. Graphene itself has metal-like zero band gap nature and it is a conducting material without further band gap engineering15,17. Therefore, the classical photoelectric effect is unlikely to contribute to the artefact, as graphene has a relatively high work function (4.5 eV). According to the experiments in this study, the amplitude level of the artefact was similar in both graphene and platinum electrodes (Fig. 2d), suggesting a similar underlying mechanism. Additional experiments also showed similar dependency on the light stimulation time and power as to the metal electrode case (Supplementary Fig. S10) ( http://www.openoptogenetics.org/index.php?title=Light-Induced_Artifact.)38. The amplitude of the artefact increased as the stimulation time was increased from 3 to 25 ms, which is similar to what has been reported previously in varying metal electrodes ( http://www.openoptogenetics.org/index.php?title=Light-Induced_Artifact.). The peak of the artefact also increased temporally as stimulation duration increased. In addition, the light power dependency was similar to that of the metal electrode38, where the artefact decreased with decreasing stimulation power (Supplementary Fig. S10b). It is interesting to note that the artefact is significantly lowered when the stimulus power are reduced, which is expected from traditional metal electrode behaviour. It is therefore important to note that the stimulus artefact may be reduced or eliminated with lower-level light stimulus, such as would be subjected during traditional imaging paradigms. Other strategies can also be employed to further characterize and reduce the stimulus artefact, as have been proposed in other optogenetic studies39, and ones that have been similarly successful in dealing with electrical stimulation artefacts40. Further study will be necessary to fully characterize the overall performance of the graphene devices and source of potential artefacts.

In vivo imaging

A subset of the implanted animals was imaged via the cranial window imaging method previously described by Schendel et al.7 Representative images of the cortical vasculature through the CLEAR micro-ECoG device are shown in Fig. 5a–d. Images in the left column were taken in bright field, while those on the right were taken under blue (470 nm) light with the aid of a tail vein injection of fluorescein isothiocyanate–dextran to fluorescently label the vasculature. These images demonstrate the clarity of the graphene electrode sites and the ability to view the underlying cortex and cerebral vasculature through the CLEAR device. Blood flow movie recordings taken through the transparent graphene electrode sites can be seen in Supplementary Movies 1 and 2. Figure 5e,f show cranial window images of a platinum micro-ECoG array, with the electrode sites and traces clearly visible.

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 data7,50. Full size image

In addition to fluorescence imaging of the cortical vasculature, optical coherence tomography (OCT) imaging demonstrates the ability of the CLEAR device based on its high transparency in the infrared spectral range. The structure of the cerebral vasculature can be captured as a three-dimensional (3D) OCT angiogram41,42,43 through the device, as shown in Fig. 6a,c. Furthermore, two typical velocity profiles44,45,46,47 of blood flow below the CLEAR device are demonstrated in Fig. 6b,d. In these images, dark lines produced by the gold traces are visible on the right side; however, the centre of the array is clear with no dark lines and vessels are easily visible. Figure 6e shows a typical cross-sectional angiogram after contrast enhancement under the CLEAR device. The OCT system is able to detect the vessels under electrode sites and electrode traces. The 3D visualization48 of the angiography data is presented in Fig. 6f. The structural data is shown in grey and vessels are shown in red. Similar data for the platinum device is shown in Fig. 6g–j, indicating the opaqueness of the platinum electrode sites. The 3D structural movies for CLEAR and the platinum devices can be seen in Supplementary Movies 3 and 4, respectively. The comparison shows the advantage of the CLEAR device over the conventional platinum device. Moreover, it is important to note that OCT uses infrared light wavelengths, which may create artefacts with ITO-based devices, depending on the fabrication process.