Dehydration and clearing of adult Drosophila for ultramicroscopy

This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, Drosophila melanogaster adults are fixed, dehydrated in ethanol, and then cleared in a solution of benzyl alcohol and benzyl benzoate.