REALM: AO-based localization microscopy deep in complex tissue

Performing Single-Molecule Localization Microscopy (SMLM) in complex biological tissues, where sample-induced aberrations hamper detection and localization, has remained a challenge. Here we establish REALM (Robust and Effective Adaptive Optics in Localization Microscopy), which corrects aberrations of ≤1 rad RMS using 297 frames of blinking molecules to improve single-molecule localization. We demonstrate this method by resolving the periodic cytoskeleton of the axon initial segment at 50 μm depth in brain tissue.