Recent advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-gram, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had onboard photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer-4 and layer-6 is differentially modulated by lit and dark conditions during free exploration.

The data in this repository was collected using the 3-photon head-mounted microscope that we present here and a calibrated setup of overhead cameras, tracking a set of head-mounted LEDs for accurate position and attitude data of the animal's head. We present raw data from the microscope and processed microscope (frame registration, denoising) and head tracking (led detection and fitting of a head-led stage model) data.

Raw data from the microscopes is included in multipage TIF format as written by ScanImage software. Multipage TIF images can be opened with many tools, e.g. ImageJ.

We have further included the post-processed data (frame registration, denoising) used in the figures in the main manuscript and the software for reproducing said figures. A detailed description of each figure is included in the README.txt file.