High‐Speed High‐Resolution Transport of Intensity Diffraction Tomography with Bi‐Plane Parallel Detection

Abstract A novel high‐speed, high‐resolution 3D microscopy technique named BP‐TIDT is presented that quantifies the refractive index (RI) distribution of label‐free, transparent samples. This method combines a bi‐plane detection scheme (BP) with the transport of intensity diffraction tomography (TIDT), effectively circumventing the need for matched illumination conditions under high numerical aperture (NA) objectives, which enables 15 fps volume rates and 326 nm lateral resolution. The effectiveness and accuracy of the proposed approach are validated through high‐resolution imaging of polystyrene microspheres and HepG2 cells. Moreover, the wide‐ranging applicability of BP‐TIDT is demonstrated by investigating subcellular organelle motion, including mitochondria and lipid droplets, as well as the macroscopic apoptosis process in living COS‐7 cells. To the best of current knowledge, this is the first time that high spatial‐temporal resolution dynamic ODT results are obtained in a non‐interferometric and motion‐free manner, highlighting the potential of BP‐TIDT in advancing research on dynamic cellular processes.