Saline-free preparation for chronic in vivo imaging in adult Drosophila

Summary Longitudinal brain imaging is essential for understanding neural mechanisms. Here, we present a saline-free, chronic preparation for repeated neural recording in adult Drosophila over multiple days. We describe steps for mounting flies, performing manual surgery on the head cuticle without external saline, and resealing the opening to create a transparent optical window. We demonstrate the utility of this approach by tracking single-neuron spiking and neuronal calcium dynamics over 7–10 days. This protocol is potentially applicable to other insect species. Graphical abstract