Exploring living zebrafish with high-speed phase-contrast CT: detailed access without contrast agents

Zebrafish have emerged as valuable animal models for studying a wide range of human diseases. The ability to visualize living zebrafish noninvasively is essential for advancing research in this field, as it enables the observation of internal tissues without compromising the organism's integrity. In this study, we assessed the feasibility of label-free imaging of living zebrafish using high-speed phase-contrast CT (PCCT). Our PCCT system integrates three critical components: a high-speed detector, a rapid-rotation sample stage, and a fast-response shutter. The detector captured data at 4000 frames per second (fps), and the stage rotated at 720 °/s. The shutter completed each opening or closing cycle within 3 ms. For each CT scan, 1000 projections were acquired along with 10 flat-field and 10 dark-field images, enabling a full 180° scan in 250 ms. Phase information was retrieved from the projections using a phase-attenuation duality (PAD)-based phase retrieval (PR) method. Tissue visibility was assessed based on the signal-to-noise ratio (SNR). For the lens, brain, and gill, the SNRs of PR-based PCCT were measured at 12.78 ± 0.47, 2.67 ± 0.39, and 1.85 ± 0.34, respectively. High-speed PCCT enabled clear visualization of zebrafish bones and soft tissues—including the eyes, brain, and gills—without the need for contrast agents, and also allowed accurate dimensional measurements of these structures in reconstructed sections. Importantly, all zebrafish survived the scanning process and resumed free swimming afterward, demonstrating the favorable biocompatibility of the technique. These results establish high-speed PCCT as a promising platform for the noninvasive, label-free investigation of zebrafish disease models, enabling detailed phenotypic observation in biomedical research.