This protocol describes the live-imaging steps of bacterial colonization of the plant root in the EcoFAB-N chamber published in the study Kuang et al., "N-dependent dynamics of root growth and nitrate and ammonium uptake are altered by bacteria Herbaspirillum seropedicae in cereal model Brachypodium distachyon" The Eco-FAB-N was imaged under an inverted time-lapse microscope after removal from the plastic enclosure that ensures sterility, and therefore although bacteria and roots can be imaged over time, this procedure is invasive.