Optogenetics combines genetic technology with optical methods to achieve high-speed and accurate regulation of neurons in living animals, cells, and tissues. Fiber photometry is a sensitive and simple method to stimulate and record neuronal activity in the deep brains of animals. Multimode fibers can transmit stimulus light to neurons and collect their fluorescence. However, some animal behaviors result from the comprehensive action of the entire brain neural network, and the small size of the multimode fiber end face limits large-scale neuroscience research. Currently, techniques that can be used to evaluate large-scale animal neurodynamics are still limited. The previously proposed multi-channel optogenetic systems face three possible problems: a low degree of freedom of parameter adjustment, the need for independent customization of multi-channel fibers, and system integration problems caused by multi-sensors. In this paper, we report a multi-channel optogenetic system. The number, order, frequency, duty cycle, and other parameters of the multi-channel fibers can be independently adjusted by targeting specific fiber channels with a galvanometer. The system includes 1-to-7 fan-out multimode fiber bundles and only a scientific complementary metal-oxide-semiconductor (sCMOS) camera as the optical sensor, which effectively reduces the difficulty of the system integration. It provides a multi-channel, independent, flexible, and highly integrated solution for multi-channel optogenetic experiments.
In this study, 1-to-7 fan-out multimode fiber bundles are used. First, a scanning galvanometer is used to target a selected fiber channel, and the time-division multiplexing technology is used to modulate lasers with different wavelengths. Subsequently, a beam with a selected wavelength is coupled to a selected fiber channel for the experiments. The fluorescence signal collected by the fiber channel is then imaged by the sCMOS camera to achieve multi-channel fluorescence recording. System debugging, instrument control, and data processing are then integrated into an automatic software system to simplify the operation process of the system and meet the requirements of the multi-channel, multiwavelength, and multifunctional optogenetics experiments. In the next step, stability and crosstalk experiments are carried out on the multi-channel optogenetic system to evaluate the uniformity and independence of the multi-channel fibers. Additionally, typical biological experiments are performed to prove the feasibility of the system in optogenetic experiments.
The multi-channel optogenetic system has excellent parameter accuracy. The scanning galvanometer can accurately target the selected beam to a selected 200-µm diameter fiber channel. The test results of the fiber output frequency show that the average error of the stimulation frequency is 0.1% within the range of 5-500 Hz. Affected by the scanning angle of the galvanometer and the uniformity of illumination, the output powers of 7 channels are slightly different, but it still has excellent power stability, and the fluctuation does not exceed 6.02% (Fig. 3). The multi-channel crosstalk experiment results show that the strong stimulation light does not interfere with the fluorescence signals collected by other channels, and the fluorescence crosstalk between channels can be ignored (Fig. 4). Therefore, each channel of the multi-channel optogenetic system has excellent anti-interference capability, which helps control noise and ensure the accuracy of the experimental results. Finally, compared to the control mice, the photostimulation of the target region can induce unilateral rotation in mice, and the statistical results show more than twice the difference (Fig. 5). The experimental results show that the power value of every channel of the system is the same. Considering the microsecond response time of the scanning galvanometer, this system has the potential in special application scenarios such as multi-channel alternate outputs and ultrafast channel scanning.
In this study, a multi-channel optogenetic system is designed, which includes a laser time-division multiplexing module, galvanometer scanning module, and 1-to-7 fan-out multimode fiber bundles. The system solves problems in previous multi-channel systems: inflexible parameter adjustment, difficult customization of multi-channel fibers, and difficult system integration caused by a large number of instruments. The system can be used in the dynamics at multiple sites across the mammalian brain. The galvanometer scans different fiber channels at a frequency of 1 kHz. The output power of a single fiber can be flexibly adjusted within the range of 0-40 mW. Additionally, this system can flexibly adjust the wavelength, frequency, duty cycle, channel number, and channel sequence. Every channel has high frequency accuracy and power stability. The software system that integrates the control, algorithm, and analysis also significantly reduces the difficulty of system operation, which provides a convenient tool for research on the functional organization and behavior-related dynamics of mesoscale circuits in the brain. In the future, a correction function can be added to realize the real-time detection of physical parameters and closed loop-automatic experiments during operation, and the weight burden of the multi-channel fiber on the head of mice can be reduced by combining lightweight technology, providing a more miniaturized and intelligent solution for the multi-channel optogenetics field.
