Comprehensive 3D Imaging of Whole Zebrafish Using a Water-Based Clearing Reagent for Hard Tissues

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ABSTRACT Zebrafish (Danio rerio) is a valuable model organism for studying developmental processes due to its external development and the optical clarity of its embryos and larvae. However, as development proceeds zebrafish form increasingly opaque tissues that impede visualization of deep tissues and structures. Although tissue clearing methods have been applied to facilitate imaging at these later stages, most of these methods have limited ability to clear dense tissues such as bone and cartilage, cause significant morphological distortion, and/or result in loss of fluorescent signal when used for imaging of fluorescent transgenes, dye stained animals, or specimens generated using immunofluorescence or fluorescence in situ hybridization methods. Here, we report a novel imaging technique using a recently developed clearing reagent called LUCID that makes it possible to capture the complete cellular-resolution 3D structures of larval and juvenile zebrafish. We show that LUCID clears dense tissues such as pharyngeal cartilage in juvenile animals and even tooth bone in adults without causing either significant morphological distortion or significant loss of signal from transgene-driven fluorescent proteins, fluorescent nuclear DNA or actin staining dyes, or whole mount in situ hybridization chain reaction (HCR) fluorescence. Using this new approach it is possible to perform complete high-resolution 3D imaging of whole fluorescently stained animals, even deep internal regions, providing a novel tool for elucidating the complex internal structures of developing zebrafish.

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