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Analysis dataset from: Simultaneous dual-color calcium imaging in freely-behaving mice

Dong, Zhe1; Feng, Yu1; Diego, Keziah1; Baggetta, Austin1; Sweis, Brian1; Pennington, Zachary1; Lamsifer, Sophia1; Zaki, Yosif1; Sangiuliano, Federico2; Philipsberg, Paul1; Morales-Rodriguez, Denisse1; Kircher, Daniel1; Slesinger, Paul1; Shuman, Tristan1; Aharoni, Daniel2; Cai, Denise1

Published Jun 19, 2025 on Dryad. https://doi.org/10.5061/dryad.fbg79cp5k

Data files

Jun 19, 2025 version files 6.49 GB

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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.