Imagining the Voltage of Neurons Distributed Across Entire Brains of Larval Zebrafish

Abstract

Neurons interact in networks distributed throughout the brain. Although much effort has focused on whole-brain calcium imaging, recent advances in genetically encoded voltage indicators (GEVIs) raise the possibility of imaging voltage of neurons distributed across brains. However, due to the high imaging speed and signal-to-noise ratio requirements of GEVIs, microscopy hardware to date has only been able to image the voltage of neurons within subregions of the brain, even for small animals like the larval zebrafish. To address this challenge, this thesis presents a high-speed remote scanning light-sheet microscope capable of imaging GEVI-expressing neurons distributed throughout entire brains of larval zebrafish at a volumetric rate of 200.8 Hz. The microscope combines remote refocusing and an ultrafast dual-camera system to significantly enhance the scanning and acquisition speed of light-sheet microscopy. Using this microscope, we measured voltage of ~1/3 of the neurons of the larval zebrafish brain, distributed throughout. We observed that neurons firing at different times during a sequence were located at different brain locations, for sequences elicited by a visual stimulus, which mapped onto locations throughout the optic tectum, as well as during stimulus-independent bursts, which mapped onto locations in the cerebellum and medulla. Whole-brain voltage imaging may open up frontiers in the fundamental operation of neural systems.