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.
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.
To collect data from throughout the brain, Eric utilized the tile-scan functionality provided by the