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[Preprint]. 2024 Jul 5:2024.07.03.601770. [Version 1] doi: 10.1101/2024.07.03.601770

Simultaneous dual-color calcium imaging 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
PMCID: PMC11244962  PMID: 39005306

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.

Teaser

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

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