This study demonstrates the noninvasive imaging of glycans in live developing zebrafish embryos using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar, peracetylated N-azidoacetylgalactosamine (Ac4GalNAz), to metabolically label their cell-surface glycans with azides. The embryos were then reacted with fluorophore conjugates via copper-free click chemistry, enabling the visualization of glycans at subcellular resolution during development. At 60 hours post-fertilization, an increase in de novo glycan biosynthesis was observed in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, the researchers performed a spatiotemporal analysis of glycan expression and trafficking, identifying patterns that would be undetectable with conventional molecular imaging approaches. This approach provides a rich source of information on the cell's physiological state, reflecting changes in glycan structures during development and disease progression.This study demonstrates the noninvasive imaging of glycans in live developing zebrafish embryos using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar, peracetylated N-azidoacetylgalactosamine (Ac4GalNAz), to metabolically label their cell-surface glycans with azides. The embryos were then reacted with fluorophore conjugates via copper-free click chemistry, enabling the visualization of glycans at subcellular resolution during development. At 60 hours post-fertilization, an increase in de novo glycan biosynthesis was observed in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, the researchers performed a spatiotemporal analysis of glycan expression and trafficking, identifying patterns that would be undetectable with conventional molecular imaging approaches. This approach provides a rich source of information on the cell's physiological state, reflecting changes in glycan structures during development and disease progression.