Single-Objective Light-Sheet Microscopy Allows Long-Term, High-Resolution In Vivo Brain Imaging of Drosophila

Abstract

In vivo imaging of dynamic subcellular brain structures is key to understanding several phenomena in neuroscience. However, in the fruit fly, its implementation has been hindered by a trade-off among spatial resolution, speed, photobleaching, phototoxicity, and setup complexity required to access specific target regions of the small brain of Drosophila. Here, we present DrosoScope: 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 (s-LNvs) in intact adult flies. We imaged the plasma membrane, cytoplasm, mitochondria, and dense-core vesicles with high spatial resolution of up to 310 nm, lower photobleaching than confocal microscopy, lower invasiveness and reduced sample-mounting complexity than alternative light-sheet technologies, and without relying on potentially phototoxic pulsed infrared lasers. Furthermore, we implemented active focus stabilization and sample-drift correction. Combined, these features enabled time-lapse brain imaging in living flies for up to 5 h, revealing neuronal plasticity and dynamic mitochondrial transport.