Visualizing Biomolecules within Living Microglia in Complex Environments Using a Clickable Small Fluorogenic Compound

Microglia, the brain's resident macrophages, are key players in neurodegenerative disease progression. While understanding how microglial organelles and biomolecules dynamically respond to physiological and pathological cues is essential for developing therapeutic strategies, current tools lack the specificity required to visualize these processes in complex, physiologically relevant environments. Here, we present CDr20-CO1, a dual-functional fluorescent probe designed for live-cell imaging of microglial biomolecules. CDr20-CO1 combines selective enzymatic activation by the microglia-specific enzyme UGT1A7C with a bio-orthogonal clickable moiety for conjugation to azide-tagged biomolecules. Using this system, we successfully visualized microglial lysosomes, phospholipids, and newly synthesized proteins in live heterogeneous brain cell cultures. Notably, this probe revealed that microglia in mixed cultures exhibited attenuated lysosomal remodeling and phosphatidylcholine metabolism in response to inflammatory stimuli compared to isolated microglia, primarily through direct cell-cell contact with neighboring glial cells, with an additional contribution from their secreted factors, underscoring the influence of the cellular microenvironment. Furthermore, CDr20-CO1 enabled in vivo labeling of microglial targets in intact live mouse embryos, demonstrating its robust tissue permeability and physiological applicability. This versatile platform provides a powerful tool for tracking microglial organelles and biomolecules in real time, offering new insights into microglial functions in health and disease.