(A) Choline derivatives for labeling phosphocholine-modified teichoic acids in S. pneumoniae. (B) Imaging of S. pneumoniae teichoic acids with propargyl-Cho (90). S. pneumoniae at different growth phases (i–iii represent early to late growth stages) was incubated in the presence of propargyl-Cho (90) for a 30 min pulse, subjected to CuAAC with azido-coumarin, and imaged by fluorescence microscopy. Left, phase contrast; right, fluorescence. White triangles mark the septal plane. Scale bars, 1 μm. Reproduced with permission from ref 251. Copyright 2017 Royal Society of Chemistry. (C) Simultaneous imaging of S. pneumoniae teichoic acids and PG with propargyl-Cho (90) and HADA (17), respectively. S. pneumoniae was incubated in the presence of propargyl-Cho (80), cyclooctyne-594, and HADA (17) for a 5 min pulse, washed, and imaged by fluorescence microscopy. Top (red), teichoic acids; middle, phase contrast; bottom (blue), PG. E, elongation site; D, division site; N, new pole; O, old pole. Right-hand boxes show fluorescence intensity as a function of cell length. Reproduced with permission from ref 253. Copyright 2018 American Chemical Society.