PURPOSE: To describe basal human tear components in negative staining with real-time transmission electron microscopy and identify extracellular vesicles using immunogold labeling. METHODS: Basal tears of 13 healthy human subjects were collected using microcapillary glass tubes, negatively stained, and observed by transmission electron microscopy. Anti-CD63 immunogold labeling was also performed to confirm the nature of elements morphologically suggestive of extracellular vesicles. RESULTS: Human tears were rich in cup-shaped vesicular structures, identified as extracellular vesicles by immunogold labeling. These vesicles were highly heterogeneous in morphology and size. Filamentous structures 200 to 700nm in length, presumed to be structures composed of phospholipids or mucopolysaccharides, were also observed. CONCLUSIONS: Two main structures were observed in human basal tears of healthy subjects, mostly extracellular vesicles. To improve applicability and translation from the laboratory bench to the hospital bedside, we provide a quick and feasible workflow for real-time analysis of human tears using transmission electron microscopy. Analysis of vesicle richness and morphology in tears could help for detecting biomarkers of ocular and systemic disease.