Improving collection efficiency in two-photon endoscopy with reflective waveguiding

Two-photon endoscopy based on a gradient-index lens has been widely utilized for studying cellular behaviors in deep-lying tissues with minimal invasiveness in vivo. Although the efficient collection of emitted light is critical to attain high-contrast spatiotemporal information, the intrinsic low numerical aperture of the endoscopic probe poses a physical limitation. We report a simple solution to overcome this limit by incorporating a reflective waveguide ensheathing the endoscopic probe, which improves the collection efficiency by approximately two-fold. We describe its principle, fabrication procedure, optical characterization, and utilities in biological tissues.