Super-Resolution Microscopy of the Bacterial Cell Wall Labeled by Fluorescent D-Amino Acids

Fluorescent D-amino acids (FDAAs) enable in situ visualization of bacterial cell wall synthesis via their incorporation into peptidoglycan (PG) crosslinks. When combined with super-resolution microscopy, FDAAs allow the details of cell wall synthesis to be resolved beyond the diffraction limit of visible light. Here, we describe using the super-resolution method of single-molecule localization microscopy (SMLM) in conjunction with two newly synthesized FDAAs (sCy₅DA and sCy₅DL_{_amide}) to resolve bacterial PG at the nanoscale in a variety of species, including Gram-negative, Gram-positive, and mycobacteria.