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.

Cells and tissues often display pronounced spatial and dynamical metabolic heterogeneity. Prevalent glucose-imaging techniques report glucose uptake or catabolism activity, yet do not trace the functional utilization of glucose-derived anabolic products. Here, we report a microscopy technique for the optical imaging, via the spectral tracing of deuterium (referred to as STRIDE), of diverse macromolecules derived from glucose. Based on stimulated-Raman-scattering imaging, STRIDE visualizes the metabolic dynamics of newly synthesized macromolecules, such as DNA, protein, lipids and glycogen, via the enrichment and distinct spectra of carbon–deuterium bonds transferred from the deuterated glucose precursor. STRIDE can also use spectral differences derived from different glucose isotopologues to visualize temporally separated glucose populations in a pulse–chase manner. We also show that STRIDE can be used to image glucose metabolism in many mouse tissues, including tumours, the brain, the intestine and the liver, at a detection limit of 10 mM of carbon–deuterium bonds. STRIDE provides a high-resolution and chemically informative assessment of glucose anabolic utilization.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Mapping the localization of multiple proteins in their native three-dimensional (3D) context would be useful across many areas of biomedicine, but multiplexed fluorescence imaging has limited intrinsic multiplexing capability, and most methods for increasing multiplexity can only be applied to thin samples (<100 µm). Here, we harness the narrow spectrum of Raman spectroscopy and introduce Raman dye imaging and tissue clearing (RADIANT), an optical method that is capable of imaging multiple targets in thick samples in one shot. We expanded the range of suitable bioorthogonal Raman dyes and developed a tissue-clearing strategy for them (Raman 3D imaging of solvent-cleared organs (rDISCO)). We applied RADIANT to image up to 11 targets in millimeter-thick brain slices, extending the imaging depth 10-to 100-fold compared to prior multiplexed protein imaging methods. We showcased the utility of RADIANT in extracting systems information, including region-specific correlation networks and their topology in cerebellum development. RADIANT will facilitate the exploration of the intricate 3D protein interactions in complex systems.

Gene expression in response to stimuli underlies many fundamental processes. However, how transcription is regulated under these scenarios is largely unknown. Here, we find a previously unknown role of nuclear actin in transcriptional regulation. The RNA-seq data reveal that nuclear actin is required for the serum-induced transcriptional program. Using super-resolution imaging, we found a remarkable enhancement of RNA polymerase II (Pol II) clustering upon serum stimulation, and this enhancement requires nuclear actin. Pol II clusters colocalized with the serum-response genes and nuclear actin filaments upon serum stimulation. Furthermore, N-WASP is required for serum-enhanced Pol II clustering. N-WASP phase-separated with Pol II and nuclear actin. In addition to serum stimulation, nuclear actin also enhanced Pol II clustering upon interferon-γ treatment. Together, our work unveils that nuclear actin promotes the formation of transcription factory on inducible genes, acting as a general mechanism underlying the rapid response to environmental cues.