Single Objective Light Sheet Microscopy allows high-resolution in vivo brain imaging of Drosophila.

In vivo imaging of dynamic sub-cellular brain structures in Drosophila melanogaster is key to understanding several phenomena in neuroscience. However, its implementation has been hindered by a trade-off between spatial resolution, speed, photobleaching, phototoxicity, and setup complexity required to access the specific target regions of the small brain of Drosophila. Here, we present a single objective light-sheet microscope, customized for in vivo imaging of adult flies and optimized for maximum resolution. With it, we imaged the axonal projections of small lateral ventral neurons (known as s-LNvs) in intact adult flies. We imaged the plasma membrane, mitochondria, and dense-core vesicles with high spatial resolution up to 370 nm, ten times lower photobleaching than confocal microscopy, lower invasiveness and complexity in sample mounting than alternative light-sheet technologies, and without relying on phototoxic pulsed infrared lasers. This unique set of features paves the way for new long-term, dynamic studies in the brains of living flies.