Abstract
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.
Highlights
Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes
Through systematic investigation of the carbon–deuterium (C–D) vibrational spectrum, we discovered Raman shifts associated with C–D bond-containing lipids, proteins, and DNA, respectively, and further revealed that this spectral selectivity resulted from the sparse labeling pattern and inherently different chemical environments surrounding the C–D bond in different types of macromolecules
We found that inhibiting protein synthesis with anisomycin in D2O-treated HeLa cells led to a peak centered at 2135 cm−1, representing the remaining D-labeled lipid signal, and blocking lipid synthesis with fatty acid synthase inhibitor TVB-3166 led to a peak centered at 2185 cm−1, representing the remaining D-labeled protein signal (Fig. 2b)
Summary
Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Compared to spontaneous Raman spectroscopy, SRS microscopy is an emerging nonlinear Raman imaging technology with substantial sensitivity boost through quantum amplification by stimulated emission, which enables at least three orders of magnitude faster acquisition time, fine spectral resolution, compatibility with fluorescence, and threedimensional (3D) optical sectioning capability in tissues and even living animals[11,12,13,14] These unique advantages of SRS microscopy, combined with our new discoveries of the chemical features of the C–D vibrational spectrum (described below), led to the development of DO-SRS microscopy, which uses D2O as a imaging contrast agent to trace lipid, protein, and DNA metabolism in cells and tissues. The separation between the O–D peak and the C–D peak means that C–D signal is essentially free of interference from the overwhelming O–D background, washing off the D2O probe before imaging is unnecessary
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