Focus-tunable two-photon fiberscope enabling in vivo imaging at selected depths

Miniaturized two-photon (2P) imaging devices enable real-time in vivo and in situ 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 ex vivo imaging of fluorescently stained convallaria and GFP mouse brain sections, as well as in vivo dynamic GCaMP-based calcium imaging of cortical neurons in an awake mouse.