Label-free phase microscopy for morphogenetic screening of zebrafish brain

Abstract

Zebrafish are emerging specimens in morphogenesis studies, due to their genetic similarity to humans, fast development, embryonic transparency, and the availability of rapid phenotypic screening. Monitoring the phenotype in zebrafish conventionally involves a visual assessment and scoring of morphological features. As advanced microscopic imaging techniques are introduced for high resolution and volumetric anatomy, these techniques have been initially applied to neuroscience and developmental studies. However, existing methods are time-consuming and methodologically limited to provide fast and morphogenetic information of whole-mount zebrafish due to shallow imaging depth and the availability of transgenic reagents. In this study, we propose a novel label-free phase microscope for morphogenetic screening of the brain of zebrafish embryos in several development stages. Our system is based on quantitative phase imaging (QPI) which recovers phase delays after light passes through the specimen. Among the various QPI methods, we used four asymmetric NIR illumination patterns providing improved contrast and penetration depth. Using clear embryo samples within 1% agarose gel, prepared by 1-phenyl 2-thiourea (PTU) treatment, we acquired the dorsal and lateral views at 3, 4, and 5 days post fertilization (dpf). As a result, we confirmed that QPI had sufficient contrast and imaging depth to identify brain regions in dorsal and lateral QPIs than bright-field images. Lateral QPIs clearly showed detailed brain structures of normal zebrafish embryos, such as epiphysis, optic tectum, and cerebellum. To further evaluate our imaging system, we compared wile-type (WT) zebrafish embryos to the atad5a mutants at 3 and 5dpf. We segmented specific parts within the brain and found that there is significant differences in size and morphology between WT and mutant animals. The midbrain of atad5a mutants became smaller and flatter than WT controls from 3 dpf and the morphological appearance of the optic tectum showed visible alteration in the mutants at 5 dpf. In conclusion, we demonstrated that our phase microscope has sufficiently high contrast and depth penetration to visualize the developmental morphology of the zebrafish brain without any labeling or contrast agents. These results showed the possibility of applying our imaging system to the screening of brain morphological alterations by genetic modification or drug treatment. Finally, the imaging capability of our system can be enhanced when it is combined with the 360-degree rotation of the specimen, which provides a quantitative, volumetric, and comprehensive brain of zebrafish developmental studies.