Simultaneous two-color imaging with a dual-channel miniscope in freely behaving mice

• Published in: Science advances Vol. 11; no. 27; p. eadr6470
• Main Authors: Dong, Zhe, Feng, Yu, Diego, Keziah, Baggetta, Austin M., Sweis, Brian M., Pennington, Zachary T., Lamsifer, Sophia I., Zaki, Yosif, Sangiuliano, Federico, Philipsberg, Paul A., Morales-Rodriguez, Denisse, Kircher, Daniel, Slesinger, Paul, Shuman, Tristan, Aharoni, Daniel, Cai, Denise J.
• Format: Journal Article
• Language: English
• Published: United States American Association for the Advancement of Science 04.07.2025
• Subjects: Animals; Behavior, Animal; CA1 Region, Hippocampal - diagnostic imaging; CA1 Region, Hippocampal - metabolism; Calcium - metabolism; Cellular Neuroscience; Hippocampus; Mice; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Neurons - metabolism; Neuroscience; SciAdv r-articles
• ISSN: 2375-2548; 2375-2548
• DOI: 10.1126/sciadv.adr6470

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. An open-source dual-channel miniscope that simultaneously records two wavelengths in freely behaving animals was presented.