4.7 Article

Optical Clearing and Light Sheet Microscopy Imaging of Amphioxus

Journal

Publisher

FRONTIERS MEDIA SA
DOI: 10.3389/fcell.2021.702986

Keywords

amphioxus; light sheet microscopy; clearing technique; whole mount immunohistochemistry; photoreceptor; acetylated tubulin; melanopsin

Funding

  1. Czech Science Foundation [20-25377S]

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Cephalochordates serve as representatives of the most basally divergent group of the chordate phylum, offering crucial insights into vertebrate evolution. In evolutionary developmental research, the combination of light sheet microscopy and tissue clearing methods allows for visualization of anatomical structures and individual cells of amphioxus in 3D space.
Cephalochordates (amphioxi or lancelets) are representatives of the most basally divergent group of the chordate phylum. Studies of amphioxus development and anatomy hence provide a key insight into vertebrate evolution. More widespread use of amphioxus in the evo-devo field would be greatly facilitated by expanding the methodological toolbox available in this model system. For example, evo-devo research on amphioxus requires deep understanding of animal anatomy. Although conventional confocal microscopy can visualize transparent amphioxus embryos and early larvae, the imaging of later developmental stages is problematic because of the size and opaqueness of the animal. Here, we show that light sheet microscopy combined with tissue clearing methods enables exploration of large amphioxus specimens while keeping the surface and the internal structures intact. We took advantage of the phenomenon of autofluorescence of amphioxus larva to highlight anatomical details. In order to investigate molecular markers at the single-cell level, we performed antibody-based immunodetection of melanopsin and acetylated-alpha-tubulin to label rhabdomeric photoreceptors and the neuronal scaffold. Our approach that combines light sheet microscopy with the clearing protocol, autofluorescence properties of amphioxus, and antibody immunodetection allows visualizing anatomical structures and even individual cells in the 3D space of the entire animal body.

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