Figure 3.

Strategies for universal biomolecule imaging in expansion microscopy (ExM). (A) Click-ExM.³⁸ Schematic of Click-ExM chemistry (top left), where cells metabolically labeled with alkynes are reacted with azide-biotin via click chemistry and stained using streptavidin-dye, then processed with the standard ExM workflow. Example data (right) of phospholipids (top, magenta), sialoglycans (middle, red), and small molecules (bottom, red) of cells treated with alkyne-choline, N-azidoacetylmannosamine (ManNAz), and azide-afatinaib, respectively. Scale bars: 10 μm (top, bottom), 2 μm (top, bottom inset), 50 μm (middle), 5 μm (middle, inset). Reproduced with permission from ref 38. Copyright 2020 Sun et al. (B) Multifunctional linking with trivalent anchoring (TRITON).³⁹ Schematic of the multifunctional probe design (top left) and an example TRITON probe (bottom left) consisting of a fluorescent reporter conjugated with a methacryloyl group and a reactive tetrafluorophenyl (TFP) ester for conjugation with amines such as B-phalloidin (for actin staining) or 1,2-distearoyl-sn-glycero-3-phos-phoethanolamine (DSPE) for lipid labeling. Example data (right) using TRITON probes for imaging cellular phospholipid membranes (pre-expansion top; post-expansion middle) through lipid conjugation to a fluorescent trivalent linker (Pacific Blue, DSPE) and α-tubulin stained via direct grafting of a DNA-conjugated secondary antibody and fluorescent oligo-based readout (post-expansion, bottom). Scale bars: 25 μm (top, middle), 5 μm (zoomed region top), 10 μm (zoomed region middle, bottom) and 5 μm (zoomed region bottom). Reproduced from ref 39. Copyright 2020 American Chemical Society.