Solvent Dehydration and Low Temperature Vacuum Drying for SEM Imaging of Pre-Hatching Frog Embryos

Scanning electron microscopy (SEM) is a fundamental technique to study the morphology of anuran embryos and tadpoles. Here, we present a drying method for SEM imaging of late frog embryos using commonly available dehydration solvents such as ethanol or methanol, xylene, and applying low temperature vacuum freeze drying. Briefly, embryos from early embryonic gills development to hatching were fixed with a paraformaldehyde-glutaraldehyde mix, then dehydrated to ethanol or methanol, and then slowly dried using low temperature and constant vacuum pressure. An extra step of clearing using xylene after ethanol dehydration improved results considerably. Our protocol successfully preserved embryo shape and the morphology of fragile and delicate superficial structures (e.g., external embryonic gills, apical ectodermal microridges and surface ciliation), while avoiding the use of some SEM toxic reagents.