Precompensation of 3D field distortions in remote focus two-photon microscopy

Remote focusing is widely used in 3D two-photon microscopy and 3D photostimulation because it enables fast axial scanning without moving the objective lens or specimen. However, due to the design constraints of microscope optics, remote focus units are often located in non-telecentric positions in the optical path, leading to significant depth-dependent 3D field distortions in the imaging volume. To address this limitation, we characterized 3D field distortions arising from non-telecentric remote focusing and present a method for distortion precompensation. We demonstrate its applicability for a 3D two-photon microscope that uses an acousto-optic lens (AOL) for remote focusing and scanning. We show that the distortion precompensation method improves the pointing precision of the AOL microscope to < 0.5 µm throughout the 400 × 400 × 400 µm imaging volume.