Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 μm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation. [Display omitted] •Scanning light-field microscopy enables 3D subcellular imaging at the millisecond scale•DAO facilitates tiled aberration corrections at high speed with a compact system•Tomographic imaging permits long-term fast intravital imaging with low phototoxicity•Migrasome biogenesis and dynamics in mammals were observed in detail by DAOSLIMIT Ultrahigh-resolution fluorescence imaging with low phototoxicity enables 3D visualization of fast cellular and subcellular processes in organoids, zebrafish, and mammals.