Four simultaneously recorded video streams of worm 3 are shown as the animal crawls freely. The location of the neurons, calcium activity, and animal behavior are all visible. The animal expresses RFP and GCaMP6s in the nuclei of neurons. The top two panels show red- and green-channel fluorescent images (respectively) that are recorded through the 40× objective. The objective translates through the volume of the worm tracing the path of a triangle wave in z with frequency 3 Hz. As the objective scans through the worm’s brain the fluorescent images go from dark (outside the worm) to bright (inside the worm) to dark again (outside the worm) giving the appearance of a 6-Hz flicker. The locations of individual neuronal nuclei are visible in the upper left panel (field of view is 150 μm wide). Fluorescence from the calcium indicator GCaMP6s is visible in the upper right panel (false color: blue is low, yellow is high; field of view is 150 μm wide). The lower two panels show the animal’s behavior imaged through the 10× objective. The collective fluorescence of all of the neurons in the animal’s head is visible in the lower right panel (field of view is 1.3 mm). This fluorescent image is used for real-time feedback to keep the head centered within the field of view. Dark-field images of the animal’s posture and behavior are visible in the lower left panel. A blue line showing the animal’s centerline is added to the images.