Spatiotemporal-multiplexed Fourier ptychographic diffraction tomography for high-speed, label-free 3D imaging of live cells

Label-free, three-dimensional (3D) imaging of organelle dynamics at high spatiotemporal resolution is essential for deciphering cellular physiology. While optical diffraction tomography (ODT) can retrieve intrinsic-contrast 3D refractive-index distribution of transparent cells, its sequential, variable-angle acquisition compromises temporal resolution, obscuring rapid events such as membrane blebbing, vesicle trafficking, and cytoskeletal remodeling. Here, we introduce spatiotemporal-multiplexed Fourier ptychographic diffraction tomography (STM-FPDT), a noninterferometric ODT modality that employs hybrid coherent/partially coherent annular illumination to achieve high resolution in both space (x−y−z) and time (t). By integrating a spatiotemporal reconstruction framework with a sliding window protocol and nonlinear global optimization, STM-FPDT achieves volumetric imaging at 5 Hz with 347 nm lateral and 1.54 μm axial resolution over a 40×/0.75 NA objective. We validate STM-FPDT across diverse cell lines, successfully capturing membrane blebbing, mitochondrial fission, and intercellular interactions that evade conventional ODT. Readily retrofittable to standard bright-field microscopes, STM-FPDT offers a broadly accessible platform for quantitative, high-spatiotemporal-resolution studies of live-cell physiology.