High-speed 3D imaging with a 25-camera multifocus microscope

High-speed volumetric imaging of whole-organism dynamics is often constrained by trade-offs between speed, resolution, and imaging depth. We present the M25 microscope, a 25-camera-array, aberration-corrected refocusing multifocus imaging system that captures 3D volumes simultaneously across 25 focal planes using a synchronized array of machine-vision cameras. Each camera incorporates a custom-blazed grating to correct chromatic dispersion, enabling a simplified, sensitive, and scalable multifocus setup for large fields of view while maintaining high spatial resolution across the imaging volume. M25 achieves imaging speeds of >100 volumes per second over imaging volumes of 180 × 180 × 50 µm. This method enables both noninvasive, label-free brightfield and highly sensitive fluorescence imaging. We demonstrate its capabilities in 3D particle tracking, fluorescent and brightfield imaging of D. melanogaster larval dynamics, and C. elegans locomotion and neural activity. This method enables fast and sensitive 3D imaging for biological studies and has potential applications across a broad range of diffractive imaging modalities.