Focus-tunable two-photon fiberscope enabling <i>in vivo</i> imaging at selected depths

Miniaturized two-photon (2P) imaging devices enable real-time <i>in vivo</i> and <i>in situ</i> imaging at subcellular resolution, highly valuable for clinical applications and basic research (such as neuroscience). However, achieving high-quality volumetric imaging at varying depths remains challenging. In this study, we demonstrated a 2P fiberscope capable of three-dimensional (3D) imaging over a cylindrical volume of a 350 μm diameter and a 400 μm depth. Depth scanning was achieved by incorporating a miniature electrowetting-based varioptic lens (VL) into a two-dimensional (2D) scanning 2P fiberscope, whose focus was tuned by modulating the VL drive voltage. The performance of the fiberscope was demonstrated by <i>ex vivo</i> imaging of fluorescently stained convallaria and GFP mouse brain sections, as well as <i>in vivo</i> dynamic GCaMP-based calcium imaging of cortical neurons in an awake mouse.