Simultaneous two-color imaging with a dual-channel miniscope in freely behaving mice
- Zhe Dong
- Yu Feng
- Keziah Diego
- Austin M. Baggetta
- Brian M. Sweis
- Zachary T. Pennington
- Sophia I. Lamsifer
- Yosif Zaki
- Federico Sangiuliano
- Paul A. Philipsberg
- Denisse Morales-Rodriguez
- Daniel Kircher
- Paul Slesinger
- Tristan Shuman
- Daniel Aharoni
- Denise J. Cai
2025-07-02
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.