Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D₂O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D₂O-derived deuterium into macromolecules generates carbon–deuterium (C–D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C–D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C–D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.

Significance: Membranes can adopt distinct phases. The endoplasmic reticulum (ER) is the largest membrane system inside cells and also harbors the richest metabolic activity including lipid synthesis. Unlike plasma membrane where separated “lipid raft” domains have been predicted and observed, ER membrane is thought to be uniformly fluidic. However, such understanding is based on biophysical studies of model membrane under thermodynamic equilibrium. It remains unclear whether and how lipid synthesis activity perturbs the equilibrium and promotes phase segregation in ER membrane. Herein, we utilized coherent Raman imaging technique to track lipid synthesis and surprisingly revealed solid-like domains emerging from liquid ER membrane. Interestingly, this phenomenon can be tuned by the incoming nutrient source, demonstrating the susceptibility of ER membrane to nonequilibrium modulation.

Significance: Cells form structures and perform functions through intricate 3D tissue organizations. However, due to tissue scattering, coherent Raman microscopy—a powerful method complementary to fluorescence imaging—suffers from limited imaging depth in tissues. Here, we develop a volumetric chemical imaging method with greater than 10-fold depth increase. We formulate a Raman-tailored tissue-clearing recipe and combine it with advanced Raman microscopies. Equipped with the toolbox of volumetric chemical imaging and analyses, we elucidate complex 3D structures, chemical compositions, and metabolic dynamics in diverse tissues including lipid synthesis throughout tumor spheroids, 3D networks of axons, vasculatures, and cell bodies in brain regions, as well as heterogeneous tumor structures and tumor metabolism.