High-resolution volumetric intravital imaging reveals asymmetric serotonin-dependent post-shock activity in the Drosophila brain

Volumetric intravital imaging of adult Drosophila brains remains challenging due to optical scattering and speed limitations of conventional microscopy. Here, we present V-shape Bessel-beam light-sheet microscopy (vSPIM), an upright, high-speed platform achieving subcellular resolution across large, opaque volumes in vivo. Through real-time calcium imaging in adult Drosophila , vSPIM uncovered two unprecedented phenomena: first, a complex ensemble of approximately 150 olfactory projection neuron boutons (more than 400 per hemisphere) resolved 18 distinct odor valence coding patterns within the dense mushroom body calyx, aligning with connectomic architecture. Second, vSPIM revealed a previously uncharacterized asymmetric, serotonin-dependent post-shock firing (PsF) response. This PsF activity emerged within the mushroom body and dopaminergic neurons only after repetitive aversive stimulation, distinct from canonical dopamine-driven reinforcement signals. Finally, we demonstrated the versatility of vSPIM by tracking corneal endothelial dynamics during mouse wound healing. Overall, vSPIM establishes a scalable framework for intravital imaging, bridging the gap between optical innovation and circuit physiology in adult tissues.