sCy5DA, a fluorescent D-amino acid, is designed for annotating peptidoglycans in live bacteria, facilitating the imaging of a broad spectrum of bacterial types, including gram-positive, gram-negative, and mycobacteria, with notably efficient incorporation into gram-negative C. crescentus. This compound allows the bacteria it labels to be visualized through single molecule localization microscopy (SMLM; known as PALM or STORM), with excitation/emission maxima reported at 646/665 nm.