Two-photon synthetic aperture microscopy for minimally invasive fast 3D imaging of native subcellular behaviors in deep tissue.

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.