Project Summary Visualizing metabolic activities in situ is critical to understanding many biological processes. It is the metabolism which carries out the genetic blue print and maintain life. Sugar such as glucose is an essential nutrient for human being and animals, not only provides energy, but also is building blocks for synthesizing new biomass such as protein, lipid, RNA/DNA, and glycogen. Our proposed study is to develop and optimize a new generation of deuterated glucose ([D]-glucose) probed 3D volumetric multiplex stimulated Raman scattering (SRS) imaging platform. In aim I, various types of [D]-glucose including [D1], [D2], [D5], [D7], and [D12]- glucose will be studied and optimized for visualizing metabolic dynamics and heterogeneity in animals. The carbon-deuterium (C-D) chemical bond in the newly synthesized molecules such as C-D labeled proteins, lipids, DNA/RNA, carbohydrates will be imaged in the “cell silent region” of Raman spectra. In addition, based on diverse spectral patterns of metabolites derived from different isotopologues, metabolite synthesis in multiple time points will be visualized in a single image. In Aim II, a new generation of enhanced tissue clearing recipe will be screened and designed for large scale 3D volumetric imaging (20x deeper penetration) of [D]-glucose probed metabolic activities and heterogeneity in animal models. In Aim III, custom designed spectral unmixing algorithm and protocol will be established to maximize the potential of detecting more than 20 newly synthesized C-D labeled molecules and minimize the background signal. Our computational resources and algorithms will optimize numerous variables in complicated hyperspectral imaging datasets for studying organ specific metabolic dynamics and heterogeneity. Using the resources and algorithms, we will optimize a platform to unmix multiple old and newly synthesized molecules and quantitatively dissect the in situ detritions. Completion of the proposed study will potentially reveal mechanistic fundamentals of multiple molecular pathways related to glucose metabolism in different organs.