Deep tissue super-resolution imaging with adaptive optical two-photon multifocal structured illumination microscopy

Visualization of axons and dendritic spines is crucial in neuroscience research.However,traditional microscopy is limited by diffraction-limited resolution and shallow imaging depth,making it difficult to study neuronal dynamics.Two-photon multifocal structured illumination microscopy(2P-MSIM)provides super-resolution imaging along with a reasonably good penetration,but it is vulnerable to optical aberrations in deep tissues.Herein we present a novel non-inertial scanning 2P-MSIM system incorporated with adaptive optics(AO)which allows for super-resolution imaging with effective aberration correction.Our strategy is designed to correct both laser and fluorescence paths simultaneously using a spatial light modulator and a deformable mirror respectively,providing better results than the individual path corrections.The successful implementation of adaptive optical two-photon multifocal structured illumination microscopy(AO 2P-MSIM)has allowed for the super-resolution imaging of neuronal structures in a mouse brain slice at great depths and dynamic morphological characteristics of zebrafish motoneurons in vivo.