Deep super-resolution imaging of thick tissue using structured illumination with adaptive optics

3D structured illumination microscopy (3D-SIM) doubles the resolution of fluorescence imaging in lateral and axial directions and increases contrast in both fixed and live specimens. However, 3D-SIM has so far not been widely applied to imaging deep in thick tissues due to its sensitivity to specimen-induced aberrations, making the method difficult to apply beyond 10 μm in depth. Furthermore, 3D-SIM has not been available in an upright configuration, limiting its use for live imaging while manipulating the specimen, for example with electrophysiology. Here, we have overcome these barriers by developing a novel upright 3D-SIM system (termed Deep3DSIM) that incorporates adaptive optics (AO) for aberration correction and remote focusing, reducing artefacts, and removing the need to move the specimen or objective. Both these advantages are equally applicable to inverted 3D-SIM microscopes. We demonstrate high-quality 3D-SIM imaging up to 130 μm into complex tissue and live sample manipulation, including human cells and Drosophila larval brains and embryos.