Two-photon fluorescence microscopy has been widely applied to three-dimensional imaging of complex samples. Remote focusing by controlling the divergence of excitation light is a common approach to scanning the focus axially. However, microscope objectives induce distortion to the wavefront of non-collimated excitation beams, leading to degraded imaging quality away from the natural focal plane. We characterized the aberrations introduced by remote focusing and used adaptive optics to correct the remote-focusing-induced aberrations. Diffraction-limited focal quality over up to 800-µm axial range can be maintained. We further demonstrated aberration-free remote focusing for in vivo imaging of neurites and synapses in mouse brain.
An enabling technology for the monitoring of neural activity, multiphoton microscopy with Bessel focus scanning is a high-speed volumetric imaging method with subcellular lateral resolution. Similar to many other optical microscopy techniques, however, its axially extended Bessel-like focus experiences sample-induced optical aberrations, which lead to reduced resolution and image contrast at depth. In this study, we demonstrated an adaptive optical Bessel focus scanning multiphoton microscope with pupil-segmentation-based wavefront sensing and highly efficient sample-plane aberration correction. Applying it to mouse brain imaging in vivo, we observed up to threefold signal enhancement for functional spine imaging at depth.
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