Abstract
Fluorescence microscopy is a method of choice for studying peptidoglycan assembly, but it presents two major challenges: the peptidoglycan must be labeled with a probe that will not perturb the physiological process, and the spatial resolution must reach the nanometer scale to reveal fine details of the synthesis process. This protocol meets both challenges by combining biorthogonal metabolic labeling of peptidoglycan in Streptococcus pneumoniae with super-resolution fluorescence microscopy (dSTORM), also providing cues to adapt it to other bacteria. For complete details on the use and execution of this protocol, please refer to Trouve et al. (2021).