Laser‐Scanning Infrared‐Raman‐Fluorescence Microscopy for Metabolic Flux Imaging in Living Organisms

ABSTRACT Comprehensive observation of chemical dynamics in living systems is crucial for deciphering metabolic processes, yet current techniques force a compromise between molecular specificity and imaging throughput. This work presents laser‐scanning in situ pump‐probe infrared (IR) and Raman excitation (LS‐INSPIRE) microscopy, a real‐time multimodal imaging platform that simultaneously captures mid‐IR photothermal (MIP), stimulated Raman scattering (SRS), and two‐photon excited fluorescence (TPEF) signals within a single scan. A custom catoptric relay paired with a reverse‐Cassegrain objective yields chromatic‐aberration‐free imaging from UV to mid‐IR, delivering pixel‐registered, multichannel data across a large field of view. The capabilities are demonstrated by tracking deuterium incorporation into lipids in live cells, quantitatively mapping molecular markers during embryogenesis, and uncovering neural development and lipid metabolism mechanisms in Caenorhabditis elegans . LS‐INSPIRE transcends the longstanding trade‐off between molecular breadth and dynamic acquisition, establishing a versatile paradigm for chemical imaging in biology, medicine, and materials science.