Neuroscientists have long debated a fundamental question about epilepsy: in which direction do seizures spread through the brain? A new study from the University of Georgia has brought fresh evidence to the debate by developing an advanced light-sheet microscope capable of fast, three-dimensional imaging of seizures in zebrafish. The researchers found that seizures originate in the hindbrain and propagate forward, or anteriorly. The results, published in Biomedical Optics Express, corroborate a previous 3D imaging study but contradict two earlier 2D imaging studies that reported propagation in the opposite direction.
«I think the discrepancies about the direction of seizure propagation point to the need for more imaging,» said Peter Kner, an engineering professor who led the research. «I believe we still don’t know how probabilistic the behaviour is and what factors are important.» The conflicting findings highlight how the choice of imaging technology can shape scientific conclusions about brain activity.
The microscope developed by Kner’s team uses a light sheet — a thin plane of light that illuminates only a single 2D section of a sample, dramatically reducing background noise from other planes. Fluorescence from the illuminated plane is captured through lenses positioned orthogonally to the light sheet and reimaged onto a camera. While light-sheet microscopy is inherently two-dimensional, the researchers extended it to 3D by dynamically sweeping the light sheet through different planes of the sample.
To keep the illuminated plane in focus, they synchronously shifted the imaging system’s focal plane using an electrically tunable lens, which changes focal length with applied current. This approach, however, introduced optical aberrations that degraded image quality. The team compensated for these distortions using a deformable mirror whose shape was carefully calibrated at each axial plane imaged.
With predetermined settings for the deformable mirror, the researchers imaged volumes of 499 x 499 x 148 micrometers at a rate of four volumes per second. They reported a fivefold increase in the area over which imaging remained near-diffraction-limited compared with uncorrected microscope images. These capabilities allowed them to observe seizure propagation in zebrafish in real time.
«This work adds pragmatic utility to 3D light-sheet microscopy for fast volumetric dynamics,» said Sixian You, an electrical engineering professor at the Massachusetts Institute of Technology who was not involved in the research. While deformable mirrors have been used to correct aberrations from tunable lenses before, You emphasized that the advance comes from pre-calibrating the corrections, which enables the fast, wide field-of-view imaging. «I expect the same approach will transfer readily to other scanning modalities that stand to benefit from tunable lenses,» she added.
After imaging the zebrafish continuously for 2.5 minutes, the researchers observed that seizures propagated from the back of the brain to the front and subsided over tens of seconds. The findings add a data point toward understanding the statistics of seizure propagation, which, if better understood, could aid epilepsy treatments in the future. Kner’s team hopes to extend the work by exploring different strains of zebrafish.
