High-speed 4D fluorescence light field tomography of whole freely moving organisms

Volumetric fluorescence imaging techniques, such as confocal, multiphoton, light sheet, and light field microscopy, have become indispensable tools across a wide range of cellular, developmental, and neurobiological applications. However, it is difficult to scale such techniques to the large 3D fields of view (FOV), volume rates, and synchronicity requirements for high-resolution 4D imaging of freely behaving organisms. Here, we present reflective Fourier light field computed tomography (ReFLeCT), a high-speed volumetric fluorescence computational imaging technique. ReFLeCT synchronously captures entire tomograms of multiple unrestrained, unanesthetized model organisms across multi-millimeter 3D FOVs at 120 volumes per second. In particular, we applied ReFLeCT to reconstruct 4D videos of fluorescently labeled zebrafish and <i>Drosophila</i> larvae, enabling us to study their heartbeat, fin and tail motion, gaze, jaw motion, and muscle contractions with nearly isotropic 3D resolution while they are freely moving. To our knowledge, as a novel approach for snapshot tomographic capture, ReFLeCT is a major advance toward bridging the gap between current volumetric fluorescence microscopy techniques and macroscopic behavioral imaging.