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.