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A Compact Closed-Loop Optogenetics System Based on Artifact-Free Transparent Graphene Electrodes

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A Compact Closed-Loop Optogenetics System Based on Artifact-Free Transparent Graphene Electrodes

Xin Liu et al. Front Neurosci.

Abstract

Electrophysiology is a decades-old technique widely used for monitoring activity of individual neurons and local field potentials. Optogenetics has revolutionized neuroscience studies by offering selective and fast control of targeted neurons and neuron populations. The combination of these two techniques is crucial for causal investigation of neural circuits and understanding their functional connectivity. However, electrical artifacts generated by light stimulation interfere with neural recordings and hinder the development of compact closed-loop systems for precise control of neural activity. Here, we demonstrate that transparent graphene micro-electrodes fabricated on a clear polyethylene terephthalate film eliminate the light-induced artifact problem and allow development of a compact battery-powered closed-loop optogenetics system. We extensively investigate light-induced artifacts for graphene electrodes in comparison to metal control electrodes. We then design optical stimulation module using micro-LED chips coupled to optical fibers to deliver light to intended depth for optogenetic stimulation. For artifact-free integration of graphene micro-electrode recordings with optogenetic stimulation, we design and develop a compact closed-loop system and validate it for different frequencies of interest for neural recordings. This compact closed-loop optogenetics system can be used for various applications involving optogenetic stimulation and electrophysiological recordings.

Keywords: closed-loop optogenetics; electrophysiology; graphene; light-induced artifact; multi-electrode array; neural recordings; optogenetics; transparent graphene array.

Figures

Figure 1
Figure 1
(A) The light transmission rate of different materials on PET and Kapton film for various wavelength. (B) Average impedance of arrays doped with nitric acid at different concentration. The data are the mean values and standard deviations of 16, 16, and 9 channels for the three methods respectively.
Figure 2
Figure 2
Fabrication processes of the flexible transparent graphene microelectrode array. (A) Cleansed silicon wafer; (B) PDMS adhesive layer; (C) PET film applied on PDMS layer; (D) Cr/Au sputtering; (E) metal wires patterned with UV-lithography and wet-etching; (F) graphene transferred by bubbling-method; (G) graphene contacts pattern with UV-lithography and oxygen plasma etching; (H) SU8 encapsulation; (I) array peeled off from the PDMS/silicon wafer.
Figure 3
Figure 3
The structure and characterization of the transparent graphene electrode array. (A) The structure of the array consists of a PET film substrate, gold wires, graphene contacts, and SU8 encapsulation. (B) A photo of the flexible array. The inset shows the transparency of the array. A Scanning Electron Microscope (SEM) image of one channel (C) and the whole array (D). Alignment marks are also included in (D) on the right side.
Figure 4
Figure 4
(A) Impedance distribution of all 16 electrodes of the array at 1 KHz. (B) Electrochemical impedance spectroscopy (EIS) of the 16 electrodes. (C) Cyclic voltammetry (CV) of a typical electrode of the array.
Figure 5
Figure 5
The pictures of artifact test for Au electrode with (A) LED stimulation off and (B) LED stimulation on.
Figure 6
Figure 6
Artifact amplitude of Au electrode. (A) The typical recording artifact of one Au electrode for different light power intensities under 20 ms pulse duration. (B) The amplitude of negative and positive peaks of the artifact measured for different light power intensities. The result shows the negative peak amplitude is increasing linearly with respect to the light pulse intensity. The positive peak amplitude increases at first and then goes to saturation with enhancing light intensity. (C) The typical recording artifact of one Au electrode for different duration under 54.1 mW/mm2. (D) The amplitude of negative and positive peaks of the artifact measured for different durations. The result shows the negative peak amplitude almost remains constant with respect to the light duration. However, the positive peak artifacts increase as duration gets longer and saturate for large light durations.
Figure 7
Figure 7
Power spectrum of artifacts recorded by the graphene and Au electrode. (A) The Au electrode records artifacts of 10 Hz and higher harmonic waves. (B) The graphene electrode shows no obvious artifacts corresponds to the light stimulation.
Figure 8
Figure 8
The comparison between the simulation results of the proposed circuit model and the experimental data for (A) fixed pulse width with increasing current source values and (B) fixed current source value with increasing pulse width.
Figure 9
Figure 9
Total output power is measured at the tip of two fibers of different diameters after coupling. (A) Output from both fibers coupled with TR2227 LED chip. This chip has a greater maximum voltage rating and it was tested until 3.5 V. (B) Output from both fibers coupled with DA2432 LED chip. This chip reached the maximum allowed current at a lower voltage rating and was tested until 3.3 V.
Figure 10
Figure 10
Coupling efficiency between fibers and (A) the TR2227 LED chip and (B) the DA2432 LED chip were calculated based on the data from Figure 9 and fiber dimensions. Higher efficiencies are observed at median flux ratings.
Figure 11
Figure 11
The closed-loop electrophysiology system. (A) The front side includes an Intan RHD2216 digital electrophysiology interface chip, power management chip, micro-switch, battery interface, and a 26-pin ZIF connector. (B) The back side locates the TI MSP430FR59891 microcontroller. (C) The diagram of the closed-loop system. (D) A schematic of the mice carrying the board.
Figure 12
Figure 12
Testing setup of the closed-loop system. (A) The diagram of the testing setup. A signal generator was used to apply pulses with different amplitudes and duty cycles into the PBS solution. The analog data was sampled by the closed-loop electrophysiology system and transmitted to the computer for visualization using serial communication. All the recordings were performed in a properly grounded Faraday cage. (B) A picture of the working system. (C) Typical real-time recording data for one channel on the customized software built by MATLAB GUI App designer. A train of 10 Hz 10 ms duration pulses modulated by a 2 Hz sine wave was applied to the saline and the threshold was set to 200 μV. (D) The recorded signal on the software for a train of 20 Hz 5 ms duration pulses modulated by 2 Hz sine wave. The threshold remains the same.

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