Background: The brain consumes a considerable amount of glucose relative to its size. Brain glucose is a source of energy, mainly via oxidative phosphorylation, as well as a source of building blocks for synthesising macromolecules. Little is known about the spatial distribution of this biosynthesis. Aim: To develop an imaging method of cerebral biosynthesis from the carbon backbone of glucose molecules at the nanometer scale Method: Injection of [U-13C]glucose into awake mice followed by correlated electron microscopy imaging and nanoscale secondary ion mass-spectroscopy (NanoSIMS) mapping of the13C/12C isotopic ratio in brain thin sections. Results: Large 13C signals were detected in somata, nucleoli, Golgi apparatus, lysosomes, dendrites and synapses. The 13C signal intensity exhibited a highly inhomogeneous spatial distribution. In particular, it was not the same in neurons and astrocytes. The time evolution of the 13C-enrichment was also strikingly different between subcellular compartments. Discussion & Conclusion: Given the fact that glucose, as well all other small, soluble metabolites, are lost in the sample preparation process, the observed 13C atoms are those incorporated into macromolecules such as nucleotides, proteins and lipids produced via biosynthetic pathways. The information obtained at the subcellular level gives a high-resolution, time-resolved map of the biosynthetic processes taking place in the brain.