A microscale analytical platform integrating microbial cell culture, isotopic labeling, along with visual and mass spectrometric imaging with single-cell resolution has been developed and applied in the monitoring of cellular metabolism in fungal mycelium. The method implements open chips with a two-dimensional surface pattern composed of hydrophobic and hydrophilic zones. Two hydrophilic islands are used as medium reservoirs, while the hydrophobic area constitutes the support for the growing aerial hyphae, which do not have direct contact with the medium. The first island, containing 12C 6-glucose medium, was initially inoculated with the mycelium (Neurospora crassa), and following the initial incubation period, the hyphae progressed toward the second medium island, containing an isotopically labeled substrate (13C6-glucose). The 13C atoms were gradually incorporated into cellular metabolites, which was revealed by MALDI-MS. The fate of the chitin-biosynthesis precursor, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), was monitored by recording mass spectra with characteristic isotopic patterns, which indicated the presence of various 12C/13C isotopologues. The method enabled mapping the 13C-labeled UDP-GlcNAc in fungal mycelium and recording its redistribution in hyphae, directly on the chip.