Adaptive optics via pupil ring segmentation improves spherical aberration correction for two-photon imaging of optically cleared tissues.

Optical clearing methods reduce the optical scattering of biological samples and thereby extend optical imaging penetration depth. However, refractive index mismatch between the immersion media of objectives and clearing reagents induces spherical aberration (SA), causing significant degradation of fluorescence intensity and spatial resolution. We present an adaptive optics method based on pupil ring segmentation to correct SA in optically cleared samples. Our method demonstrates superior SA correction over a modal-based adaptive optics method and restores the fluorescence intensity and resolution at high imaging depth. Moreover, the method can derive an SA correction map for the whole imaging volume based on three representative measurements. It facilitates SA correction during image acquisition without intermittent SA measurements. We applied this method in mouse brain tissues treated with different optical clearing methods. The results illustrate that the synaptic structures of neurons within 900 μm depth can be clearly resolved after SA correction.