  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 micrometer
throughout the 400 x 400 x 400 micrometer imaging volume.
