Aberration correction for deformable mirror based remote focusing enables high-accuracy whole-cell super-resolution imaging

Abstract

AbstractSingle-molecule localization microscopy (SMLM) enables three-dimensional (3D) investigation of nanoscale structures in biological samples, offering unique insights into their organization. However, traditional 3D super-resolution microscopy using high numerical aperture (NA) objectives is limited by imaging depth of field (DOF), restricting their practical application to relatively thin biological samples. Here, we developed a unified solution for thick sample super-resolution imaging using a deformable mirror (DM) which was served for fast remote focusing, optimized point spread function (PSF) engineering and accurate aberration correction. By effectively correcting the system aberrations introduced during remote focusing and sample aberrations at different imaging depths, we achieved high-accuracy, large DOF imaging of the whole-cell organelles [i.e. nuclear pore complex (NPC), microtubules, and mitochondria] with a nearly uniform resolution of approximately 30 nm across the entire cellular volume.