This study presents a method for non-invasive in vivo imaging of glycans in developing zebrafish embryos using a chemical reporter strategy. Glycans, which are complex carbohydrates attached to proteins and lipids, are challenging to image due to their incompatibility with genetically encoded reporters. The researchers used an unnatural sugar, Ac₄GalNAz, to metabolically label cell-surface glycans with azides. Subsequently, they used copper-free click chemistry with fluorophore conjugates to visualize glycans in live embryos at subcellular resolution. The method involved treating zebrafish embryos with Ac₄GalNAz, which is incorporated into mucin-type O-linked glycoproteins. The embryos were then reacted with DIFO-fluorophore conjugates to label glycans. The technique allowed for the visualization of glycans in specific regions of the embryo, such as the jaw, pectoral fins, and olfactory organs, where de novo glycan biosynthesis was observed. The study also demonstrated the ability to distinguish between newly synthesized and previously reacted glycans using two- and three-color detection experiments. This approach revealed distinct patterns of glycan expression and trafficking, which would be undetectable with conventional molecular imaging methods. The results showed that glycans are dynamically regulated during development, reflecting the activity of the cell's secretory machinery and metabolic pathways. The method was validated using zebrafish cell lines and whole-embryo lysates, showing robust metabolic labeling and identification of glycoproteins. The technique was found to be non-toxic and effective for in vivo imaging, with no developmental abnormalities observed. The study highlights the potential of this approach for imaging other biomolecules and provides insights into the dynamic regulation of glycans during zebrafish embryogenesis.This study presents a method for non-invasive in vivo imaging of glycans in developing zebrafish embryos using a chemical reporter strategy. Glycans, which are complex carbohydrates attached to proteins and lipids, are challenging to image due to their incompatibility with genetically encoded reporters. The researchers used an unnatural sugar, Ac₄GalNAz, to metabolically label cell-surface glycans with azides. Subsequently, they used copper-free click chemistry with fluorophore conjugates to visualize glycans in live embryos at subcellular resolution. The method involved treating zebrafish embryos with Ac₄GalNAz, which is incorporated into mucin-type O-linked glycoproteins. The embryos were then reacted with DIFO-fluorophore conjugates to label glycans. The technique allowed for the visualization of glycans in specific regions of the embryo, such as the jaw, pectoral fins, and olfactory organs, where de novo glycan biosynthesis was observed. The study also demonstrated the ability to distinguish between newly synthesized and previously reacted glycans using two- and three-color detection experiments. This approach revealed distinct patterns of glycan expression and trafficking, which would be undetectable with conventional molecular imaging methods. The results showed that glycans are dynamically regulated during development, reflecting the activity of the cell's secretory machinery and metabolic pathways. The method was validated using zebrafish cell lines and whole-embryo lysates, showing robust metabolic labeling and identification of glycoproteins. The technique was found to be non-toxic and effective for in vivo imaging, with no developmental abnormalities observed. The study highlights the potential of this approach for imaging other biomolecules and provides insights into the dynamic regulation of glycans during zebrafish embryogenesis.