Abstract
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.
Highlights
Two-photon scanning microscopy is a widely used tool for high resolution functional imaging in life sciences due to its ability to penetrate deep within scattering tissue [1,2]
Rapid remote focusing has been implemented with electrically tunable lenses (ETLs) [3], liquid crystal spatial light modulators (LC-SLMs) [4,5,6,7,8], phase-locked ultrasound lenses (tunable acoustic gradient (TAG) lenses) [9], deformable mirrors [10], liquid lenses [11], acousto-optic lenses (AOLs) [12,13,14,15] and a secondary objective coupled with a piston mirror [16,17,18,19,20]
Wavefront-shaping devices are increasingly being used in two-photon microscopy to provide the rapid focusing required for 3D functional imaging and 3D photostimulation
Summary
Two-photon scanning microscopy is a widely used tool for high resolution functional imaging in life sciences due to its ability to penetrate deep within scattering tissue [1,2]. Remote focus devices cannot always be positioned in a plane conjugate to the back aperture of the objective, due to mechanical space constraints, a limited range of commercially available focal length lenses and uncertainty in the exact position of the back aperture of commercial objectives This is problematic because it introduces depth-dependent 3D field distortions in the imaging volume [3,21,22,23,24], which impair the performance of remote focus laser scanning systems. As the laser is focused in z away from the natural plane (the focal plane for a planar input beam), the FOV progressively drifts laterally This is problematic as even modest misalignments of the remote focus unit can result in distortions of the dimensions of the FOV and lateral shifts amounting to tens of micrometers over the focus range. We demonstrate experimentally that a paraxial remote focus distortion model can be applied to an AOL remote focus microscope to precompensate for the 3D field distortions arising from non-telecentric misalignments and show how similar distortion precompensation solutions could be applied to other remote focus technologies
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