Functional small biomolecules play indispensable roles inside cells. However, sensitive and specific visualization of these molecules in living systems has proven to be highly challenging. Herein, we report stimulated Raman scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues, and visualizing de novo synthesis of DNA, RNA, proteomes, phospholipids and triglycerides, respectively, through metabolic incorporation of alkyne-tagged small precursors.

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon-deuterium bonds (C-D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.stable isotope labeling | stimulated Raman microscopy | protein synthesis

Cholesterol is essential for cells to grow and proliferate. While normal mammalian cells meet their need for cholesterol through its uptake or de novo synthesis 1 , the extent to which cancer cells rely on each of these pathways remains poorly understood. Here, using a competitive proliferation assay on a pooled collection of DNA-barcoded cell lines, we identified a subset that is auxotrophic for cholesterol and thus highly dependent on its uptake. Metabolic gene expression analysis pinpointed loss of squalene monooxygenase (SQLE) expression as a cause of the cholesterol auxotrophy, particularly in ALK+ anaplastic large cell lymphoma (ALCL) cell lines and primary tumors. SQLE catalyzes the oxidation of squalene to 2,3-oxidosqualene in the cholesterol synthesis pathway and its loss results in accumulation of the upstream metabolite squalene, which is normally undetectable. In ALK+ ALCLs, squalene alters the cellular lipid profile and protects cancer cells from ferroptotic cell death, providing a growth advantage under conditions of oxidative stress and in tumor xenografts. Finally, a CRISPR-based genetic screen identified cholesterol uptake by the low-density lipoprotein receptor (LDLR) as essential for the growth of ALCL cells in culture and as patient-derived xenografts. This work reveals that the cholesterol auxotrophy of ALCLs is a targetable liability, and, more broadly, that systematic approaches are useful for identifying nutrient dependencies unique to individual cancer types.

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.

Conspectus Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because that common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, a Bioorthogonal Chemical Imaging platform has recently been introduced. By coupling the stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e. g. alkynes and stable isotopes), Bioorthogonal Chemical Imaging exhibits superb sensitivity, specificity and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides/deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and to model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor Bioorthogonal Chemical Imaging, a valuable technique in the era of “omics”. As a unique tool for biological discovery, this platform has been applied to studying various metabolic processes under both physiological and pathological states, including protein synthesis activity of neuronal systems, protein aggregations in Huntington disease models, glucose uptake in tumor xenograft and drug penetration through skin tissues. We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophore has done for larger molecular species.

SignificanceMembranes 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.

A primary goal of metabolomics is to identify all biologically important metabolites. One powerful approach is liquid chromatography-high resolution mass spectrometry (LC-MS), yet most LC-MS peaks remain unidentified. Here, we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. We consider all experimentally observed ion peaks together, and assign annotations to all of them simultaneously so as to maximize a score that considers properties of peaks (known masses, retention times, MS/MS fragmentation patterns) as well network constraints that arise based on mass difference between peaks. Global optimization results in accurate peak assignment and trackable peak-peak relationships. Applying this approach to yeast and mouse data, we identify a half-dozen novel metabolites, including thiamine and taurine derivatives. Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to annotate untargeted metabolomics data, revealing novel metabolites.

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.