No abstract

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-g, 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 on-board 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.

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. We show that neuronal population activity in cortical layer-4 and layer-6 was differentially modulated by lit and dark conditions during free exploration.

Laser scanning microscopy requires beam steering through relay and focusing optics at sub-micron precision. In light-weight mobile systems, such as head mounted multiphoton microscopes, distortion and imaging plane curvature management is unpractical due to the complexity of required optic compensation. Thus, the resulting scan pattern limits anatomical fidelity and decreases analysis algorithm efficiency. Here, we present a technique that reconstructs the three-dimensional scan path only requiring translation of a simple fluorescent test probe. Our method is applicable to any type of scanning instrument with sectioning capabilities without prior assumptions regarding origin of imaging deviations. Further, we demonstrate that the obtained scan pattern allows analysis of these errors, and allows to restore anatomical accuracy relevant for complementary methods such as motion correction, further enhancing spatial registration and feature extraction.