Fig. 1.

Simultaneous imaging of whole-brain calcium activity and behavior, simplified schematic. A worm crawls freely on a motorized stage under near-infrared (NIR) dark-field illumination. A spinning disk confocal microscope acquires volumetric fluorescent images of the worm’s brain by scanning a 40× objective along the imaging axis (z) to acquire 6 brain volumes/s. A low-magnification 10× objective images the animal’s posture and behavior. Custom 3D real-time tracking software feeds back on the fluorescence images and adjusts the $xy$ motorized stage and z-piezo stage accordingly to keep the worm’s head centered in the high magnification objective’s field of view.