Simultaneous dual-color calcium imaging in freely-behaving mice

Area of Science:

• Neuroscience
• Bioengineering
• Optical Imaging

Background:

• Minimally invasive fluorescence microscopy, or miniscopes, are crucial for observing neural activity in behaving animals.
• Current miniscopes are limited to single-wavelength recordings, restricting simultaneous observation of multiple cellular processes.
• There is a need for advanced imaging tools capable of capturing concurrent signals from different fluorescent reporters.

Purpose of the Study:

• To introduce an open-source, dual-channel miniscope capable of simultaneous two-wavelength imaging in freely behaving animals.
• To validate the performance and minimize crosstalk between the two imaging channels.
• To demonstrate the utility of the dual-channel miniscope in tracking dynamic neural activity alongside static cellular markers.

Main Methods:

• Integration of two CMOS sensors into a single miniaturized microscope.
• Simultaneous acquisition of two distinct fluorescence wavelengths.
• Validation using co-expressed calcium indicators (GCaMP) and nuclear reporters (tdTomato) in the mouse hippocampus.
• Behavioral experiments involving mice running on a linear track.

Main Results:

• Successful simultaneous imaging of two fluorescence wavelengths with minimal crosstalk.
• Demonstration of stable long-term neuronal registration using the static tdTomato signal.
• Observation of dynamic changes in hippocampal spatial coding over time, evidenced by GCaMP signals.
• The dual-channel miniscope effectively captured concurrent neural and cellular information.

Conclusions:

• The novel open-source dual-channel miniscope provides a powerful tool for simultaneous multi-wavelength imaging in neuroscience.
• This technology overcomes previous limitations of single-wavelength miniscopes, enabling more complex experimental designs.
• The minimal crosstalk and robust performance open new avenues for investigating neural circuits and dynamics in behaving animals.