Glucose is one of the most fundamental molecules within life and bioengineering sciences. Present understanding of its role in cellular and bioengineering processes relies primarily on invasive, large-scale biochemical analysis, providing no spatial information on glucose pools or fluxes. This work identifies an emerging microscopy technique based on coherent anti-Stokes Raman scattering (CARS), which fulfills the need of quantitative imaging of glucose at the single-cell level with submicrometer resolution. No sample preparation with reporter molecules is required, ensuring that the low-weight metabolite is studied under natural conditions. The potential of CARS microscopy is illustrated by quantitatively mapping glucose fluxes and distributions in a microfluidic bioreactor and in lipid-bilayer vesicles, the latter as a model for glucose transmembrane transport. Furthermore, the metabolic response to a glucose pulse was monitored in living yeast cells. This study signifies a new era within CARS microscopy for its use of monitoring carbohydrates, in particular glucose which is one of the most abundant molecules in nature. Copyright © 2010 John Wiley & Sons, Ltd.