Buchmann Institute for Molecular Life Sciences 1 article published in JoVE Developmental Biology Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos Frederic Strobl1,2,3, Selina Klees1,2,3, Ernst H. K. Stelzer1,2,3 1Physical Biology, Buchmann Institute for Molecular Life Sciences (BMLS), 2Cluster of Excellence Frankfurt, Macromolecular Complexes, 3Goethe-Universität Frankfurt am Main – Campus Riedberg Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.