Simultaneous Behavioral and Neuronal Imaging by Tracking Microscopy.

Tracking microscopy enables whole-brain cellular resolution imaging in freely swimming animals. This technique enables both structural and functional imaging without immobilizing the animal, and greatly expands the range of the behaviors accessible to neuroscientists. We use infrared imaging to track the target animal in a behavioral arena. Based on the predicted trajectory of the brain, we apply optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We have combined this motion cancellation system with Differential Illumination Focal Filtering (DIFF), a form of structured illumination microscopy, which enables us to image the brain of a freely swimming larval zebrafish for over an hour. Here we describe the typical experimental procedure for data acquisition and processing using the tracking microscope.