Cell imaging maps MAGL probe engagement, not the enzyme itself

Monoacylglycerol lipase (MAGL) plays a central role in endocannabinoid signaling and represents a key therapeutic target for neurodegenerative and inflammatory diseases, cancer, and psychiatric conditions. Sensitive tools for monitoring MAGL distribution in cells are therefore essential for elucidating its biological function. Here, we report the design, synthesis, and cellular application of the covalent, alkyne-tagged MAGL probe 1, which enables enzyme-associated vibrational imaging through detection of the enzyme-bound probe by spontaneous Raman and stimulated Raman scattering (SRS) microscopy. The probe exhibits a distinct C≡C stretching band at 2210 cm-1 within the "cell-silent" region, enabling intracellular detection of the non-chromophoric enzyme across concentrations ranging from 0.5-25 μM, including robust visualization at 0.5 μM, a concentration relevant to clinically advanced MAGL inhibitors. Spontaneous Raman and SRS imaging revealed enrichment of the enzyme-bound probe at lipid-rich intracellular interfaces, consistent with lipid droplet-associated MAGL localization, while SRS enabled rapid, high-contrast subcellular mapping. Control experiments, including competitive inhibition, confirmed that the observed signal arises predominantly from the covalently bound probe 1 rather than unreacted species. Target specificity was further supported by activity-based protein profiling, excluding nonspecific lipid accumulation. Rather than directly imaging the enzyme itself, the vibrational signal reports on covalent probe-enzyme engagement as a surrogate for enzyme localization. Correlative fluorescence lifetime imaging microscopy using a matched BODIPY-labeled MAGL fluoroprobe confirmed target engagement and provided orthogonal vibrational and fluorescence readouts. These findings demonstrate the feasibility of covalent enzyme-targeted Raman imaging as a complementary modality to fluorescence-based approaches for imaging non-chromophoric enzymes in cells using matched molecular pair probes.