This video captures advanced multimodal imaging of a head-restrained mouse under very low ambient light conditions. Initially, the screen appears dark, but the mouse’s pupil becomes visible, shining white due to the infrared laser used to image neuronal activity. The laser is directed through a glass window surgically implanted in the skull, replacing a portion of the bone to provide optical access to the brain. Some photons from the laser traverse the brain and skull, becoming visible in the mouse’s eye. Key data collected include:

• Neuronal Activity: Neurons expressing the calcium indicator GCaMP6s emit green fluorescence when active (firing). The infrared laser excites these neurons through the glass window, allowing visualization of brain activity.
• Pupil Diameter: The shining pupil reflects the laser’s interaction with the brain and provides data on arousal states.
• Locomotion: The mouse is placed on a treadmill, enabling precise tracking of movement.
• Whisker Dynamics: Two infrared cameras capture whisker movements, enabling 3D pose estimation of each whisker through deep learning algorithms.
• Whisker Stimulation: Stimulation events are recorded to correlate sensory input with neuronal and behavioral responses.

This experimental setup provides a comprehensive platform to study the interplay between sensory input, neuronal activity, arousal, and motor behavior in awake mice with unprecedented precision.