Twist-free ultralight two-photon fiberscope enabling neuroimaging on freely rotating/walking mice

Lightweight and head-mountable scanning nonlinear fiberscope technologies offer an exciting opportunity for enabling mechanistic exploration of ensemble neural activities with subcellular resolution on freely behaving rodents. The tether of the fiberscope, consisting of an optical fiber and scanner drive wires, however, restricts the mouse's movement and consequently precludes free rotation and limits the freedom of walking. Here we present the first twist-free two-photon fiberscope technology for enabling neuroimaging on freely rotating/walking mice. The technology equips a scanning fiberscope with active rotational tracking and compensation capabilities through an optoelectrical commutator (OEC) to allow the animal to rotate and walk in arbitrary patterns during two-photon fluorescence (TPF) imaging of neural activities. The OEC provides excellent optical coupling stability (<±1% fluctuation during rotation) and an extremely high torque sensitivity (<8 mN · m). In addition, the new technology is equipped with a custom grating and prism to effectively manage the temporal properties of the femtosecond excitation pulses through the fiber-optic system, which improved neuroimaging signal by more than 2X. This TPF fiberscope imaging platform has been tested for in vivo imaging, and the results demonstrate that it enables reliable recording of calcium dynamics of more than 50 neurons simultaneously in the motor cortices of freely behaving mice in a twist-free fashion. With active tracking function of the OEC enabled, we observed considerable increase in both behavior and neural activities in the motor cortices of the mice during freely behaving neuroimaging experiments.