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Abstract

The miniaturized two photon (2P) microscope or ‘fiberscope’ allows imaging during free movement, requiring continuous tracking of the head and eyes to determine visual input. We developed a 2P-compatible, all-optical system for head and eye tracking in rodents. Head tracking with 6 DOF employed infrared LEDs mounted on the microscope and imaged by multiple overhead cameras, while miniaturized camera systems with specialized, custom-built optics and electronics were used to image the eyes (see accompanying poster for details). Calibration procedures based on the Tsai camera model realistically incorporated radial lens distortion, and for custom-built camera systems decentering and thin-prism distortions as well. To detect eye movements, we directly compared 3D geometric models of the eye and pupil to each observed image, minimizing an objective function over eye rotation angles and pupil dilation radii. We found that this approach, which detected the 2D pupil boundary and 3D eye rotation simultaneously in a single step, was more robust than previous methods with an intermediate stage of 2D feature detection, allowing our system to operate effectively at lower contrast. Since the pupil-iris boundary deviated slightly from a perfect circle, with an uneven, crenellated appearance on a fine spatial scale, we also detected ocular torsion by measuring rotation of this rough boundary through 3D space. The eye tracker was self-calibrating in that animals were not required to fixate a presented target, aiding the use of this system in rodents where such training is impossible. Finally, based on the appearance of the eyeball-eyelid boundary we defined anatomically based coordinate axes and baseline pupil positions that were consistent across animals, even when the location and orientation of eye tracking cameras varied. Together, these tracking systems and analysis methods allowed stimulus presentation monitors and other environmental features to be mapped continuously onto each pupil plane, and gaze vectors for each eye to be projected into the animal’s environment.