Superresolution imaging of Drosophila tissues using expansion microscopy.

The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.