Microscopy is a key technique to visualize and understand biology. Electron microscopy (EM) facilitates the investigation of cellular ultrastructure at biomolecular resolution. Cellular EM was recently revolutionized by automation and digitalisation allowing routine capture of large areas and volumes at nanoscale resolution. Analysis, however, is hampered by the greyscale nature of electron images and their large data volume, often requiring laborious manual annotation. Here we demonstrate unsupervised and automated extraction of structures and biomolecules in conventionally processed tissues using large-scale hyperspectral energy-dispersive X-ray (EDX) imaging. First, we discriminated biological features in the context of tissue based on selected elemental maps. Next, we designed a data-driven workflow based on dimensionality reduction and spectral mixture analysis, allowing the visualization and isolation of subcellular features with minimal manual intervention. Broad implementations of the presented methodology will accelerate the understanding of biological ultrastructure.