Scientists have captured the rapid spread of seizures across a living zebrafish brain in three dimensions, thanks to an ultra-fast light sheet fluorescence microscopy system developed at the University of Georgia.
Light sheet microscopy has been a powerful tool for studying the zebrafish central nervous system. This technique shines a thin sheet of laser light through a single layer of a sample at a time, reducing light damage, or photobleaching, and increasing imaging speed.
Speed is essential when capturing the hundreds of neurons firing within milliseconds in the brain. This is why light sheet microscopy is often combined with volumetric imaging, which images thin slices through the depth of a zebrafish brain and stacks them to create a three-dimensional volume that captures each neuron’s position and activity.
In a paper in Biomedical Optics Express, researchers used existing three-dimensional light sheet microscopy techniques to build a microscope fast enough to capture zebrafish brain activity during a seizure.
Previously, researchers at the University of Georgia built a light sheet microscopy design that could capture three-dimensional volumetric images with a large field of view. But the system was too slow to capture seizure activity, taking about 1.75 seconds to acquire a single raw volume.
To improve the new microscope design’s speed, researchers modified the previous electrically tunable lens (ETL), which keeps the image in focus by shifting the focal plane up and down, to run continuously with synchronized sinusoidal waveforms. This means the lens moves smoothly through the depth of the zebrafish brain without stopping for each image slice, boosting imaging speed to seven times that of the previous design.
Changes in imaging speed and ETL movement can add distortion or blurriness to the final image. To address these concerns, the researchers reprogrammed the system to use sensorless adaptive optics technology to correct optical aberrations and keep the final image precise and sharp, preserving image quality while running faster.
The final light sheet system with an ETL and reprogrammed adaptive optics captured zebrafish volumes up to 499 × 499 × 150 µm3 at 4 volumes per second with high resolution, enabling seizure propagation tracking. At this speed and resolution, they exposed zebrafish larvae to a known convulsant agent and monitored calcium waves in the brain.
Sources: Biomedical Optics Express, 2026., Biomedical Optics Express, 2023.