Noninvasive, label-free visualization of living killifish embryos using high-speed phase-contrast CT

• High-speed PCCT enables rapid, noninvasive, and label-free visualization of living killifish embryos with high visibility for both mineralized and soft tissues. • Quantitative safety assessment confirms that embryos scanned via PCCT develop normally, with hatching success and locomotor activity comparable to unexposed controls. • The PCCT system combines four key advantages: full noninvasiveness, no requirement for contrast agents, exceptional structural visibility, and preservation of embryonic viability, offering strong potential for developmental biology research. Noninvasive, label-free visualization of living embryos represents a notable advancement in developmental sciences. In this study, we aimed to evaluate the capability of a high-speed phase-contrast CT (PCCT) system for noninvasive, label-free visualization of living embryos. This assessment was conducted by examining both the imaging efficacy and biosafety of the system using live killifish embryos as the model organism. The PCCT system integrated three key components: a fast-acquisition detector, a rapid-rotation stage, and a precision electromechanical shutter. To assess irradiation safety, embryos were exposed to varying exposure durations, and their post-irradiation viability was evaluated based on hatching success, larval swimming behavior, and locomotor activity. These experiments quantitatively determined the threshold radiation dose that preserves embryonic development. PCCT scans were acquired using the detector operating at 1,250 frames per second (fps), while the sample stage rotated at 450 °/s. Each CT scan, comprising 500 projections over a 180° rotation, was completed in 400 milliseconds. Phase retrieval (PR) processing significantly enhanced tissue visibility, increasing the signal-to-noise ratio (SNR) from 0.82 ± 0.04 to 13.4 ± 1.4. The system provided excellent visualization for both highly absorbing structures (e.g., otoliths, vertebrae, and caudal fin) and low-attenuation soft tissues (e.g., eyes, brain, yolk sac, liver parenchyma, kidney, and intestinal tracts). Crucially, despite an absorbed irradiation dose (AID) of 1.222-1.241 Gy, all scanned embryos (10/10) developed normally with 100% viability; their hatching, morphology, and locomotion remained indistinguishable from unexposed controls, collectively confirming the system's high biosafety. Our results demonstrate that high-speed PCCT serves as an advanced imaging platform that uniquely integrates four critical advantages: complete noninvasiveness, label-free operation, superior tissue visibility, and preserved embryo viability. These advanced capabilities highlight the system's strong translational potential for embryo-related developmental biology research.