Extracellular particles, including non-vesicular extracellular particles (NVEPs) and extracellular vesicles (EVs), are emerging as key contributors to sensory signaling, yet their ultrastructural organization within native tissues remains underexplored. Native tissues preserve extracellular particle organization and heterogeneity that are often lost during dissociation. Using cryofixation-based serial block-face scanning electron microscopy, we generated a nanoscale atlas of NVEPs and EVs across 352 Drosophila antennal olfactory sensilla (~70 % coverage). Segmentation of more than 7,800 extracellular particles revealed distinct populations differing in morphology, size, electron density, and sensillum-class distribution. Analyses of EV biogenesis identified multivesicular bodies and putative membrane budding in auxiliary cells. Furthermore, sensilla housing degenerating neurons exhibited marked accumulation of EVs and NVEPs, accompanied by increased auxiliary-cell EV biogenesis. These findings provide a large-scale ultrastructural characterization of extracellular particles in native sensory tissues and establish a foundation for understanding how extracellular particles are distributed, generated, and function during sensory signaling and degeneration.

The SBEM volumes analyzed in this study were generated previously (Choy et al., 2025; Nava Gonzales et al., 2021). Briefly, 6- to 8-day-old female flies were cold-anesthetized, and their antennae were dissected. After isolation of the third antennal segment, samples were processed using high-pressure freezing, freeze substitution, rehydration, DAB labeling, en bloc heavy metal staining, dehydration, and resin infiltration. Flies expressed membrane-tethered APEX2 (10xUAS-myc-APEX2-Orco or 10xUAS544 mCD8GFP-APEX2) in select ORNs under the control of specific OrX-GAL4 drivers, as described previously (Tsang et al., 2018; Zhang et al., 2019). Resin-embedded specimens were imaged by micro computed X-ray tomography to determine sample position and orientation, then mounted on aluminum pins using conductive silver epoxy and sputter-coated with gold-palladium. SBEM imaging was performed using either a Gemini SEM (Zeiss) equipped with a 3View block-face unit or a Merlin SEM 549 (Zeiss) equipped with a 3View2XP system and OnPoint backscatter detector. After data collection, the images were converted to MRC format, and rigid alignment of the image slices was performed using cross-correlation in the IMOD image processing package.