Light-sheet microscopy is an emerging technology that enables imaging of biological specimens with minimal photo-damage. It opens new avenues to study cell biological and developmental processes with unprecedented imaging speed. It also facilitates systems biology approaches by quantitative imaging of entire living systems. Light-sheet microscopy has only recently left the optical tables of technology developers and became more broadly available through commercial and open-access platforms. This EMBO Practical Course will comprehensively introduce the light sheet microscopy paradigm to the new generation of scientists.