The Link Lab at the Medical College of Wisconsin studies ocular development and disease, focusing on zebrafish models for retinal neurogenesis. Eric Clark, a Ph.D candidate in the Link Lab, sought to create a full-volume 3D image of a whole zebrafish brain approximately 750 microns in depth, and spanning an area approximate 3 millimeters in diameter. To image the nervous system, the animal was first transfected with a construct expressing eGFP behind a pan-neuronal promoter, resulting in a ubiquitous green fluorescence throughout neurons in the CNS.
Sample clearing and objective lens
To reduce scattering caused by changes in refractive index(RI), the dissected CNS sample was cleared via passive clarity. A custom Histodenz based refractive index matching solution was used to maintain consistent RI throughout the optical path. The prepared organ was imaged using a Leica HC FLUOTAR L 25x/1.00 IMM objective lens , that offers 250 nm optical resolution in a field of ca 1mm x 1mm at up to 6 mm free working distance. This objective is equipped with a motorized correction (motCORR) collar, which can be adjusted to compensate for the effects of RI variance at depth.
Video animation of eGFP stained zebrafish
Collection of high resolved large field
To collect data from throughout the brain, Eric utilized the tile-scan functionality provided by the