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, there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes and durations due to point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in two-photon microscopy to achieve aberration-corrected 3D imaging of subcellular dynamics at millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in phototoxicity. With its unique advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in mouse lymph node, and characterized heterogenous cellular states in mouse visual cortex, opening up a new horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.Funding Information: Z.Z., Y.Z., J.H., Y.C., J.F., X.L., J.Z., Z.W., J.W., Q.D. were supported by the National Natural Science Foundation of China (62088102, 61927802, 62071272). H.Q. were funded by the National Natural Science Foundation of China (81621002).Declaration of Interests: The authors declare the following competing interests: QD, JW, ZZ, YZ, and JF had patent applications related to the 2pSAM method developed in this work.Ethics Approval Statement: All the experimental procedures were approved by the Use Committee and Institutional Animal Care at Tsinghua University, Beijing, China.