Ultra-deep penetration of temporally-focused two-photon excitation

Abstract

ABSTRACT Temporal focusing (TF) nonlinear microscopy enables simultaneous illumination of relatively large areas while maintaining optical sectioning, by relying on the sensitivity of multiphoton processes to pulse duration. Line temporal focusing (LITEF) combines temporal focusing in one plane (xz) and spatial fo cusing in the perpendicular plane (yz). The additional spatial focusing improves optical sectioning compared to wide field temporal focusing and exhibits improved performance in scattering medium. Two photon microscopy’s ultimate depth of penetration is limited by out-of-focus excitation. This work explores whether LITEF can be used to address this limitation. Here, we present experimental results displaying the feasibility of ultra-deep penetrati on two-photon excitation in scattering media (>>1mm) using LITEF without significant distortions or out-of-focus-excitation. Our experimental setup is based on an amplified 800nm ultrafast laser where a dual-prism gra ting (DPG) is used as a diff ractive element, allowing light to propagate on-axis throughout the optical setup, and providing a high diffraction efficiency. These results present new opportunities for ultra deep, optically sectioned 3D two photon imaging and stimulation within scattering biological tissue, beyond the known out-of-focus excitation limit. Keywords: Temporal Focusing, Ultra Deep, Multiphoton Microscopy, Line illumination Temporal Focusing, Two-Photon Excitation