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Transparent and Flexible Low Noise Graphene Electrodes for Simultaneous Electrophysiology and Neuroimaging

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Transparent and Flexible Low Noise Graphene Electrodes for Simultaneous Electrophysiology and Neuroimaging

Duygu Kuzum et al. Nat Commun.

Abstract

Calcium imaging is a versatile experimental approach capable of resolving single neurons with single-cell spatial resolution in the brain. Electrophysiological recordings provide high temporal, but limited spatial resolution, because of the geometrical inaccessibility of the brain. An approach that integrates the advantages of both techniques could provide new insights into functions of neural circuits. Here, we report a transparent, flexible neural electrode technology based on graphene, which enables simultaneous optical imaging and electrophysiological recording. We demonstrate that hippocampal slices can be imaged through transparent graphene electrodes by both confocal and two-photon microscopy without causing any light-induced artefacts in the electrical recordings. Graphene electrodes record high-frequency bursting activity and slow synaptic potentials that are hard to resolve by multicellular calcium imaging. This transparent electrode technology may pave the way for high spatio-temporal resolution electro-optic mapping of the dynamic neuronal activity.

Figures

Figure 1
Figure 1. Description of graphene electrodes
(a) Schematic illustration of a flexible graphene neural electrode array. Patterned graphene electrodes are in contact with Au contact pads to interface with the data acquisition system. (b) Photograph of a 16-electrode transparent array. The electrode size is 300 × 300 µm2. Inset, a closer view showing the electrode area. Fainted squares are the electrode openings in the encapsulation layer. (c) Microscope image of the array in b. Fainted patterns of graphene electrodes and wires are visible. (d) Microscope image of an 8-electrode hippocampal slice array. The electrode size is 50 × 50 µm2.
Figure 2
Figure 2. Electrochemical characterization of graphene electrodes
(a) EIS results for 50 × 50 µm2 Au, G and doped-graphene samples. Measurement results are shown with symbols and fitting results are shown with solid lines for impedance magnitude (top figure) and phase (bottom figure) plots. The impedance magnitude (top figure) significantly decreased with doping of graphene, more prominently for frequencies lower than 1 kHz. (b) Cyclic voltammogram showing enhanced charge storage capacity for doped-graphene electrode. Electrode size is 50 × 50 µm2. Legend shows calculated charge by integrating the area under the CV curve. (c) Schematic for the equivalent circuit model used to fit EIS measurement results. CPE is the constant phase element representing the double-layer capacitance; RCT is the charge transfer resistance; ZW is the Warburg element representing the diffusion of charges species to the interface; and RS is the solution resistance. The models for circuit elements are provided in Supplementary Materials.
Figure 3
Figure 3. In vivo neural recordings in rats
The data presented here is representative of three different acute experiments, each of them lasting 5–6 hours. (a) The photograph of a 50 × 50 µm2 single graphene electrode placed on the cortical surface of the left hemisphere and a 500 × 500 µm2 single Au electrode placed on the cortical surface of the right hemisphere. The inset shows the flexibility of the electrodes. (b) Interictal-like spiking activity recorded by 50×50 µm2 doped-graphene and Au electrodes. Both electrodes were placed on the same hemisphere and connected to the same amplifier channel in subsequent recordings. Data was filtered with a 0.1 Hz-3 kHz bandpass filter. Recordings with doped-graphene electrodes are five to six-fold less noisy compared to the ones with same size Au electrode. (c) Power density spectra of the recordings shown in b calculated over 20 s time window. Doped-graphene electrode recorded significantly lower 60 Hz noise and its harmonics. The inset shows almost two orders of magnitude decrease in 60Hz electrical interference noise. (d) Seizure-like discharges recorded by 50×50 µm2 doped-G and 500×500 µm2 Au electrodes simultaneously according to the electrode placement shown in a. Similar SNR are observed for both recordings.
Figure 4
Figure 4. Calcium imaging with confocal and two-photon microscopy setups
(a) Optical transmission spectrum of 12.5 µm and 25 µm bare Kapton films and 25 µm Kapton film coated with graphene. The fluorescence emission range of the calcium indicator dye, Oregon Green Bapta-1 (OGB-1) is shown in green shadow. The excitation wavelength (488 nm) used in the confocal microscopy is delineated by the dotted blue line. The excitation wavelength used in two-photon microscopy (840 nm) is delineated by the dotted red line. (b) Schematic illustration of the confocal microscopy setup with a custom imaging chamber for simultaneous imaging and recording. Insert: Hippocampus slice tissue was mounted on the graphene electrode and perfused with ACSF throughout the experiment. Note that excitation as well as emission light passes through the graphene electrode. (c) Schematic illustration of the two-photon microscopy setup with a custom imaging chamber. Two-photon microscopy operates with an upright microscope equipped with a high numerical aperture water immersion lens. Insert: In order to keep the slice tissue healthy, the custom chamber is equipped with a mesh support and the solution exchange occurs at the bottom of the tissue and the top of the electrode. (d) Left schematic shows different regions of the hippocampus. The dentate gyrus is imaged by the confocal microscope. Images show Left: A steady-state fluorescence (F0) image of dentate gyrus in a OBG-1 AM stained hippocampal slice obtained through the 50×50 µm2 graphene electrode. The graphene electrode is seen as a square outline with dark edges. Middle: Simultaneously obtained transmittance image. Right: Merge of the steady-state fluorescence (right) and the transmittance images (left). Scale bar is 50 µm. (e) Dentate gyrus is imaged by the two-photon microscope. Images show Left: A steady-state fluorescence (F0) image of dentate gyrus in a OBG-1 AM stained hippocampal slice obtained through the 50×50 µm2 graphene electrode. The graphene electrode is seen as a square outline with dark edges. Middle: Simultaneously obtained transmittance image. Right: Merge of the steady-state fluorescence (right) and the transmittance images (left). Scale bar is 50 µm.
Figure 5
Figure 5. Simultaneous multi-cellular calcium imaging and electrophysiology using transparent graphene electrodes
The data presented here is representative of four different in vitro experiments performed on 12 rodent hippocampal slices. (a) Left: A steady-state fluorescence (F0) image of the dentate gyrus in a OBG-1 AM stained hippocampal slice obtained by confocal microscopy. The excitation light at 488nm as well as the fluorescence emission (max at ~520nm) penetrated through the transparent electrode (graphene and polyimide substrate). Middle: Simultaneously obtained transmittance image. Right: Region of interest (ROI) for electrode area and randomly selected six cells within the electrode area are outlined. Calcium transients from these cells are shown in Fig. 5c. Bottom: Color-coded images of normalized fluorescence change (ΔF/F0) at baseline (0s) and during one of the peak responses (7.08s). Scale bar is 50 µm. (b) Recording from the graphene electrode shows interictal-like activity, i.e., short population bursts (shown in insert) that last less than a second, occurring every 5–12 seconds during the 2 minutes of recording. (c) The calcium transient (ΔF/F0) for the electrode area (labeled as ROI) shows an increase in calcium signal coinciding with the interictal-like event recorded in (b). ΔF/F0 traces for individual cells show that most of the cells responded with the interictal-like event but with varied amplitude. Cell 2 did not respond to the third event. Cell 3 only responded to the forth event with a delayed peak. See Supplementary Movie 1. (d) Left: A steady-state fluorescence (F0) image of a different part of the dentate gyrus. Middle: Simultaneously obtained transmittance image. Right: ROI for electrode area and randomly selected six cells within the electrode area are outlined. Bottom: Color-coded images of normalized fluorescence change (ΔF/F0) at baseline (0s) and during one of the peak responses (9.41s). Scale bar is 50 µm. (e) Recording from the graphene electrode shows ictal (seizure)-like activity, i.e., prolonged population bursts (shown in insert) that lasted ~10sec, occurring 3 times during ~150 seconds of recording. (f) Calcium transients (ΔF/F0) for the electrode area (labeled ROI) increased to an elevated level (plateau) with respect to baseline during the ictal-like events. During the ictal-like event, cells 1,2,4, and 5 exhibited plateaus while and cells 3 and, 5 showed multiple peaks. Cell 6 did not participate in ictal-like activity. See Supplementary Movie 2.
Figure 6
Figure 6. Corrosion protection and inhibition of electrochemical reactions by graphene:
(a) Cyclic voltammogram for Au and graphene coated Au (G/Au) electrodes. G/Au sample does not exhibit any faradaic reaction peaks, showing that graphene acts as a protective layer covering Au surface. The electrode size is 500×500 µm2(b) Raman spectroscopy measured on the same G/Au samples dipped it in PBS. Measurements were taken before dipping in PBS, after 11 days, after 30 days and after 6 months in PBS.

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