Repeated, noninvasive, high resolution spectral domain optical coherence tomography imaging of zebrafish embryos

Purpose: To demonstrate a new imaging method for high resolution spectral domain optical coherence tomography (SDOCT) for small animal developmental imaging. Methods: Wildtype zebrafish that were 24, 48, 72, and 120 h post fertilization (hpf) and nok gene mutant (48 hpf) embryos were imaged in vivo. Three additional embryos were imaged twice, once at 72 hpf and again at 120 hpf. Images of the developing eye, brain, heart, whole body, proximal yolk sac, distal yolk sac, and tail were acquired. Three-dimensional OCT data sets (501x180 axial scans) were obtained as well as oversampled frames (8,100 axial scans) and repeated line scans (180 repeated frames). Scan volumes ranged from 750x750 μm to 3x3 mm, each 1.8 mm thick. Three-dimenstional data sets allowed construction of C-mode slabs of the embryo. Results: SD-OCT provided ultra-high resolution visualization of the eye, brain, heart, ear, and spine of the developing embryo as early as 24 hpf, and allowed development to be documented in each of these organ systems in consecutive sessions. Repeated line scanning with averaging optimized the visualization of static and dynamic structures contained in SD-OCT images. Structural defects caused by a mutation in the nok gene were readily observed as impeded ocular development, and enlarged pericardial cavities. Conclusions: SD-OCT allowed noninvasive, in vivo, ultra-high resolution, high-speed imaging of zebrafish embryos in their native state. The ability to measure structural and functional features repeatedly on the same specimen, without the need to sacrifice, promises to be a powerful tool in small animal developmental imaging.