Despite evidence linking arterial calcification to acute cardiovascular event risk, the physicochemical mechanisms underlying mineral nucleation and growth in atherosclerotic plaques remain unknown. Prospective clinical data show that the risk of cardiovascular events inversely correlates with the density of calcification present within arterial plaques. Finite element modeling of stress distribution within plaques indicates that subcellular microcalcifications in a plaque’s fibrous cap promote material failure of the plaque, causing myocardial infarction and stroke. In contrast, large calcifications may stabilize the plaque, but mechanisms that give rise to these two morphologies are unclear. The formation of both large calcifications and microcalcifications in the atherosclerotic plaque may involve extracellular vesicles (EVs) released by cells within the plaque that serve as nucleating foci for mineralization. The lack of certainty in this process exists due to the inability to visualize calcification nucleation and growth in vivo. Using advanced high-resolution microscopic and spectroscopic analyses of calcified human atherosclerotic plaques and a three-dimensional synthetic collagen hydrogel system to mimic the plaques, we provide a crucial link between plaque collagen content and calcification morphology—the two determinants of atherosclerotic plaque stability—thus offering novel insight into the mechanisms of plaque integrity. Confocal images revealed small microcalcifications abundantly present throughout the fibrous cap of human carotid plaques obtained from endarterectomy or autopsy. The microcalcifications appeared to form in gaps between collagen fibers and are composed of discrete spheres less than 1 µm in diameter. Pearson correlation analysis revealed a significant inverse relationship between microcalcification area and the collagen content within the analyzed area (n = 9 plaques, r = -0.29, p = 0.01). Microcalcifications were also found directly adjacent to large calcific regions, where the microcalcifications appeared to merge, forming the larger calcifications. Density-dependent color scanning electron microscopy (DDC-SEM) revealed structural heterogeneity within the large calcific regions with small calcium phosphate-rich microcalcification spheres less than 1 µm in diameter aggregating to form the larger calcific region. We developed a three-dimensional in vitro preparation to visualize these processes to test our hypothesis that nucleation and growth of calcific mineral depends on calcification and aggregation of cell-derived EVs. Human coronary artery smooth muscle cells (SMCs) from primary donors were cultured in control or calcifying media for 14 days, a sufficient time for the SMCs to release specialized calcifying EVs. The EVs were collected and added to collagen hydrogels, mimicking the fibrous cap of atherosclerotic plaques. After 72 h incubation at 37 oC, the calcifying samples exhibited structures observed by confocal reflection microscopy in gaps between collagen fibers that contained regions of calcification as detected by a molecular imaging calcium tracer. DDC-SEM revealed aggregation of EVs from the calcifying media samples between collagen fibers, forming dense structures resembling those observed within calcified human plaques. Structured illumination super-resolution microscopy of fluorescently labeled EVs provided an optical approach to visualize EV aggregation and calcification. This technique allowed visualization of objects the size of individual EVs aggregating (approximately 200 nm in diameter) within the collagen hydrogels. EVs appeared to aggregate to produce spherical microcalcifications. Increasing the collagen concentration within the hydrogels from 0 to 1 mg/ml reduced the average aggregate size by 90% compared to the samples without collagen (n=4, ANOVA, p < 0.001). We provide direct visualization of vascular calcific mineral nucleation and growth using multimodal imaging and spectroscopic techniques. We show that calcification growth and maturation results from a series of aggregation processes that is controlled by collagen concentration. We found an inverse relationship between collagen content in the fibrous cap of human atherosclerotic plaques and the size of observed microcalcifications. Further, by increasing collagen concentration in three-dimensional hydrogels, we showed a decrease in the size of EV aggregates that form within the hydrogel and a concomitant decrease in the maturity of the resulting calcific mineral. These observations suggest that formation of microcalcifications accompanies thinning of the fibrous cap. We propose that inflammation leading to collagen degradation within the cap allows localized calcifying EV aggregation and microcalcification formation, thus unifying two prominent theories of plaque instability.