Our understanding of the dynamics of peptidoglycan growth has been hampered by our inability to directly image peptidoglycan synthesis in vivo. Given the role of D-amino acids in crosslinking the glycan strands, Kuru et al. investigated whether fluorescent probes attached to a D-amino acid backbone could be incorporated into peptidoglycan during its synthesis. The authors attached the small fluorophores 7-hydroxycoumarin 3-carboxylic acid (HCC-OH) and 4-chloro-7-nitrobenzofurazan (NBD-C1) to 3-amino-D-alanine (ADA) to create HADA and NADA, respectively. Strong peripheral and septal labelling of the entire population of viable bacteria was observed with both fluorophores for phylogenetically diverse bacterial species, including Escherichia coli, Agrobacterium tumefaciens and Bacillus subtilis. Furthermore, pulse–chase experiments allowed real-time tracking of peptidoglycan synthesis using time-lapse microscopy.