Denitrification, a key process in soil nitrogen cycling, occurs predominantly within microbial hotspots, where denitrifiers use nitrate as an alternative electron acceptor. For accurate prediction of dinitrogen (N2) and nitrous oxide (N2O) emissions from denitrification, a precise quantification of these microscale hotspots is required. Employing a unique combination of X-ray CT imaging, microscale O2measurements, and15N labeling, we were able to quantify hotspots of aerobic respiration and denitrification. We analyzed the dynamics of greenhouse gas (GHG) fluxes, soil oxygen supply, and the distribution of particulate organic matter (POM) in intact soil samples from a grassland and a cropland under different moisture conditions. Our findings reveal that both free and occluded particulate organic matter (POM), identified through X-ray CT imaging, contribute to GHG emissions. The occluded POM, i.e. POM at distant locations to air-filled pores, emerged as a primary driver of denitrification within structured soils of both land uses. Thus, the higher denitrification rates in the grassland could be attributed to the higher content of occluded POM. Conversely, despite possessing compacted areas that could favor denitrification, the cropland had only small amounts of occluded POM to stimulate denitrification. This underlines the complex interaction between soil structural heterogeneity, organic carbon supply, and microbial hotspot formation and thus contributes to a better understanding of soil-related GHG emissions. In summary, our study provides a holistic understanding of soil-borne greenhouse gas emissions and emphasizes the need to refine predictive models for soil denitrification and N2O emissions by incorporating the microscale distribution of POM.