Afg3022c

Electrical stimulation of cell culture was performed on the 3D-CNFs with a self-made bioreactor as showed in Fig. 1b. Isolated cells were seeded into 3D-CNFs at 5 × 10⁵ cells per well. After 24 h in culture, stainless steel electrodes were inserted to form the electrical contact with the 3D scaffolds. A 100 Hz pulsed electrical field of 100 mV cm⁻¹ was applied across the two electrodes for 4 h in the incubator (at 37 °C with 5% CO₂) with a function generator (AFG3022C, Tektronix, USA) daily for one week. Cells were analysed 24 h after electrical stimulation.

The ultrasonic stimulation system and parameters are the same as our previous studies (18 (link)). In the LITUS system, two generators (AFG3022C; Tektronix, USA) were used to generate the pulse signal. After amplification by a linear power amplifier (E&I240 L; ENI Inc., USA), the pulse signal is transmitted to the unfocused ultrasonic transducer (V301-SU; Olympus, Japan). After collimating through a 10 mm diameter collimator, ultrasound was applied to the damaged area of the rat (Figure 1A). Figure 1B shows the time and intensity parameters of ultrasound. The total stimulation duration was 10 min with 200 trials and the duration of each trial is 3 s. The ultrasound fundamental frequency (FF) and pulsed repetition frequency (PRF) were 500 kHz and 1 kHz, respectively. The tone-burst duration (TBD) and stimulation duration (SD) were 0.5 ms and 400 ms, respectively. The Isppa value was 2.6 W/cm² (Figure 1B). During LITUS stimulation, the Non-invasive Vital Signs Monitor (V1.0, Shanghai Yuyan Instruments Co., Ltd.) was applied to monitor heart rate, respiration and blood pressure of rats to reflect humanistic care. Prior to each LITUS stimulation, anesthesia was performed by intraperitoneal injection of 10% chloral hydrate (4 mL/kg). During the stimulation, the rats were fixed on a stereotaxic apparatus to ensure the accuracy of each stimulus position (Figure 1C).

In the LIPUS system, two connected function generators (AFG3022C; Tektronix, United States) were used to generate the pulsed signals. The pulsed signal from the second generator was amplified using a linear power amplifier (E&I240 L; ENI Inc., United States) and transmitted to an unfocused ultrasound transducer (V301-SU; Olympus, Japan). Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (3%, 5 mg/100 g, IP). The ultrasound transducer was applied to the designated region in the injured cortical areas using a conical collimator with a diameter of 10 mm and was filled with ultrasound coupling gel. The total stimulation duration was 10 min. LIPUS was administered immediately to the rats and the rats were treated with LIPUS stimulation once per day for 7 d. The ultrasound fundamental frequency (FF) and pulsed repetition frequency (PRF) were 500 and 1 kHz, respectively. The ultrasound stimulation duration (SD) and tone-burst duration (TBD) were 400 and 0.5 ms, respectively (Li et al., 2017 (link)). The ultrasound pressure was measured using a calibrated needle-type hydrophone (HNR500; Onda, United States), and the spatial peak and pulse-average intensity (Isppa) was 2.6 W/cm2. The method is similar to our previous study (Wu et al., 2020 (link)).

The pulsed signal generated by two arbitrarily connected function generators (AFG3022C, Tektronix, USA) was amplified by a linear power amplifier. An unfocused ultrasound transducer with a fundamental frequency of 500 kHz (V301-SU, Olympus, USA) was used in our experiment (Fig. 1). In addition, a 3D printed conical collimator filled with US coupling gel was used to connect the mouse head with the transducer to reduce the absorption and distortion of the sound waves at a 45° angle to the electrodes. The stimulation duration, pulse repetition frequency and duty cycle were set as 50 ms, 1 Hz, and 5%, respectively. The ultrasound pressure under the skull measured by a calibrated needle-type hydrophone (HNR500; Onda, USA) in the water tank was 0.51 MPa, and its corresponding I_sppa and I_spta were 8.67 W/cm² and 0.43 W/cm². We determined the placement of the ultrasound transducer and coupling cone based on the mouse brain atlas and the distribution map of the ultrasound field [14 (link)]. This ensured that the ultrasound was targeted to the motor cortex. The total time for each stimulation was 5 min.
Fig. 1

Schematic of ultrasound stimulation and EEG and EMG recording

The materials and devices were characterized using an optical microscope (Nikon Eclipse LV100ND), an SEM (FEI Nova NanoSEM430, acceleration voltage of 1 kV), an AFM (Bruker Dimension Icon), and a UV–Vis–NIR spectroscope (Varian Cary 5000). The electrical and optoelectronic performances were measured using a semiconductor analyzer (Agilent B1500A), a probe station (Cascade M150), an input signal generator (Tektronix AFG 3022C), an oscilloscope (Tektronix MSO 2024B), and a laser diode controller (Thorlabs ITC4001, using laser excitations of 405 and 516 nm) in a dark room at room temperature. The noise was measured by a noise measurement system (PDA NC300L, 100 kHz bandwidth). With the help of special mask to avoid crosstalk issues (Supplementary Fig. 30), the electrical performance of the 1024 phototransistors in the optoelectronic sensor array was automatically measured using a home-built transistor array test system (Agilent B1500A and Keysight 34980A) controlled by a self-developed program, and the data analysis and image processing were carried out using MATLAB (Supplementary Figs. 31–33).

The low voltage tone burst signal is generated by a Tektronix AFG3022C arbitrary waveform generator and amplified using a Krohn-Hite high voltage power amplifier and the amplified voltage signals are directly measured by a Tektronix DPO4034B digital oscilloscope connected to the output terminals of operational amplifiers in NC circuits. The out of plane velocity field on the surface of the sample is measured using a Polytec PSV-200 scanning laser vibrometer. The wave propagation properties within the GRIN metamaterial are calculated based on the velocity amplitude averaged over regions extending 300 mm on either side of the metamaterial region with a spatial resolution of approximately 1.5 mm. The velocity signal is acquired with a 128 kHz sampling frequency. For each point, we measure the response of the system to 20 consecutive tone burst signals to obtain reliable data.

The experimental setup includes sound signal generation, acoustoelectric signal conversion, and signal acquisition and processing devices (as shown in fig. S10). An arbitrary function generator (AFG3022C, Tektronix) generates oscillating signals, which are then amplified by a power amplifier (5016, SAST) before driving a speaker (SUINY). At the same time, a commercial microphone (MPA231, BSWA), placed at the same distance as the MBAS near the defect cavity, is used to measure the incident sound pressure as a reference. This sensor is positioned outside of the MBAS defect cavity so that it measures the typical air pressure after propagation over the same distance as the MBAS but without sound focusing and pressure enhancement. A data acquisition system with analysis software (DHDAS, Donghua) is used to record all data from the MBAS and the microphone simultaneously. We carried out the whole test in a semianechoic chamber, with a typical background noise at 15.6 dB.