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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 an 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 (dTomato) while mice ran on a linear track. Our results suggest that, even when neurons were registered across days using dTomato signals, hippocampal spatial coding changes over time. In conclusion, our dual-channel miniscope enables imaging of two fluorescence wavelengths with minimal cross-talk between the two channels, opening the doors to a multitude of previously inaccessible experimental possibilities. 

Abstract 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. TeaserNovel open-source dual-channel Miniscope that simultaneously records two wavelengths with minimal crosstalk in freely behaving animals.