Abstract
Zebrafish are a powerful model for imaging studies of development and neurobiology. However, most studies have focused on developing zebrafish due to technical and biological challenges of imaging adult stages. These include increased tissue opacity and illumination depth limitations, and difficulty maintaining life support and anesthesia in a 2-4cm long fish. There are currently limited tools for intravital imaging of the adult zebrafish brain. The ability to image the brain in the same individual repeatedly without physical damage would allow zebrafish to be better utilized to study aging and neurodegenerative disease. We designed and applied a 3D-printable device for non-invasive, repeatable multiphoton neural imaging of genetically non-pigmented adult zebrafish from 2 months to 19 months old. Animals successfully recovered after multi-hour imaging sessions and can be imaged repeatedly over periods of weeks to years. We show the utility of this approach through imaging the neuroimmune system, revealing that microglia in aged zebrafish have enhanced cellular dynamics. This technique could be widely used and beneficial for other cell-scale neuroimaging studies in the adult fish.