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Miniaturized fluorescence microscopes (miniscopes) enable imaging of calcium events from a large population of neurons in freely behaving animals. Traditionally, miniscopes have only been able to record from a single fluorescence wavelength. Here, we present a new open-source dual-channel Miniscope that simultaneously records two wavelengths in freely behaving animals. To enable simultaneous acquisition of two fluorescent wavelengths, we incorporated two CMOS sensors into a single Miniscope. To validate our dual-channel Miniscope, we imaged hippocampal CA1 region that co-expressed a dynamic calcium indicator (GCaMP) and a static nuclear signal (tdTomato) while mice ran on a linear track. Our results suggest that, even when neurons were registered across days using tdTomato signals, hippocampal spatial coding changes over time. In conclusion, our novel dual-channel Miniscope enables imaging of two fluorescence wavelengths with minimal crosstalk between the two channels, opening the doors to a multitude of new experimental possibilities.

The human brain represents one of the most complex biological systems, containing billions of neurons interconnected through trillions of synapses. Inherent to the brain is a biochemical complexity involving ions, signaling molecules, and peptides that regulate neuronal activity and allow for short- and long-term adaptations. Large-scale and noninvasive imaging techniques, such as fMRI and EEG, have highlighted brain regions involved in specific functions and visualized connections between different brain areas. A major shortcoming, however, is the need for more information on specific cell types and neurotransmitters involved, as well as poor spatial and temporal resolution. Recent technologies have been advanced for neuronal circuit mapping and implemented in behaving model organisms to address this. Here, we highlight strategies for targeting specific neuronal subtypes, identifying, and releasing signaling molecules, controlling gene expression, and monitoring neuronal circuits in real-timein vivo. Combined, these approaches allow us to establish direct causal links from genes and molecules to the systems level and ultimately to cognitive processes.