Recently, the Miniscope project team at the University of California, Los Angeles (UCLA) has innovated based on its fourth-generation miniature microscope (version v4) and developed the world's first open-source dual-channel miniature microscope. Published in *Science Advances*, this research, by integrating dual CMOS sensors and a focus-tunable optical system, achieves dual-color synchronous imaging in freely behaving mice, opening up a new dimension for neural circuit research. ### Research Background The field of neuroscience research has long faced a crucial challenge: how to simultaneously observe multiple neural activity markers in freely moving animals. In neuroscience studies, understanding the collaborative mechanisms between different neural populations is vital for unraveling the mysteries of the brain. The synchronous observation of multiple neural activity markers can provide us with more comprehensive and in-depth information. Traditional miniature microscopes (miniscopes) have played an important role in neuroscience research, enabling calcium imaging at single-cell resolution. However, their single-channel design has obvious limitations, as they can only record signals at a single fluorescence wavelength. This prevents scientists from simultaneously obtaining information about multiple neural activity markers during research, limiting in-depth studies on the collaborative mechanisms of neural populations. With the rapid development of genetically encoded fluorescent probes, scientists' technical demands have been continuously increasing. They urgently need a technology capable of synchronously capturing multi-wavelength signals to reveal the collaborative mechanisms between different neural populations. The synchronous capture of multi-wavelength signals allows scientists to more clearly observe the interactions and collaborative relationships between different neural populations. In studying the stability of hippocampal spatial representation, synchronous imaging of dynamic calcium signals (such as GCaMP) and static cell markers (such as dTomato) is crucial. The hippocampus is significant for spatial memory and navigation, and "representational drift" is a research hotspot. Traditional methods are limited, while synchronous imaging enables scientists to accurately analyze the relationship between the two, providing strong support for deciphering the "representational drift" phenomenon and facilitating in-depth understanding of hippocampal functions and neural mechanisms. ### Technological Innovation Based on this, the research team focused on innovating and developing based on the fourth-generation miniature microscope (version v4). After unremitting efforts, they successfully developed the world's first open-source dual-channel miniature microscope, bringing new hope to neuroscience research. During the development process, the research team demonstrated excellent innovation capabilities. They innovatively integrated two PYTHON480 CMOS sensors into a miniaturized device weighing only 4.8g. This move not only achieved the miniaturization of the device but also made it possible to collect multi-wavelength signals simultaneously. Such a small device, yet with powerful functions, reflects the wisdom and exquisite skills of the researchers. To achieve effective separation and collection of signals at different wavelengths, the research team adopted a high-transmittance dichroic beamsplitter. This beamsplitter can separate the emitted light of GCaMP (green channel) and dTomato (red channel), and when combined with single-band emission filters, it controls crosstalk between channels to below 7.5%. The application of this technology ensures the accuracy and reliability of the collected signals, laying a solid foundation for subsequent data analysis. In terms of imaging, a unique sliding adjustment mechanism plays an important role. Since GRIN lenses cause a chromatic aberration shift of approximately 3.5μm, which can affect imaging quality, the sliding adjustment mechanism can effectively compensate for this deviation, ensuring dual-channel confocal imaging. Through this design, the microscope can obtain clear and accurate images, providing more intuitive research data for researchers. Compared with single-channel microscopes, this open-source dual-channel miniature microscope has significant advantages. It achieves dual-wavelength synchronous acquisition while maintaining a 1000×1000μm field of view and a 30fps frame rate. ### Experimental Verification To conduct in-depth research on neural activity, 10 C57BL/6J male mice were selected as experimental subjects, and combined with 1mm diameter GRIN lens implantation technology, preparations were made for subsequent cross-day imaging experiments. The experimental site was set on a 1-meter linear runway, and the entire imaging process lasted 13 days. In the physical experiment, the mice needed to wear the 4.8g dual-channel device during activities. Surprisingly, even with the device, the mice could reach a running speed of 45.11cm/s. This indicates that the dual-channel device has little impact on the mice's activity ability and does not interfere with their normal behavior, ensuring the accuracy of experimental data. Through seven cross-day imaging sessions, the researchers achieved important results. They successfully tracked 150 CA1 neurons, gaining a deeper understanding of the activity of these neurons. Further analysis found that among these neurons, 46.4% of dTomato-labeled cells could be stably detected throughout the experiment. This result shows the stability of dTomato labeling in long-term experiments. However, the situation is different for GCaMP-positive cells. Only 22.2% of GCaMP-positive cells could be cross-day registered through calcium signals. This data highlights the necessity of a static labeling channel, which can provide more stable labeling information and make up for the deficiency of dynamic calcium signals in cross-day registration. Finally, the researchers evaluated the crosstalk between channels through linear regression analysis. The results confirmed that the crosstalk coefficient between channels was significantly lower than the theoretical value (P < 0.001). This result indicates that the dual-channel device has high reliability in the signal acquisition process, can effectively reduce interference between channels, and provides strong support for the accuracy of experimental data.