Creager MD, et al. - Whether ¹⁸F-fluoride positron emission tomography (PET)/computed tomography (CT) can recognize early microcalcifications in atherosclerotic plaques was investigated to gain clarity regarding the underlying mechanism of signal amplification seen in PET-positive, CT-negative image regions. Researchers used an in vitro 3-dimensional hydrogel collagen platform, ex vivo human specimens, and a mouse model of atherosclerosis to validate ¹⁸F-fluoride signal amplification derived from microcalcifications against near-infrared fluorescence molecular imaging and histology. They observed an inverse correlation of microcalcification size with collagen concentration. Amplification of the PET signal by smaller microcalcifications was suggested by the finding that ¹⁸F-fluoride ligand bound to microcalcifications formed by calcifying vascular smooth muscle cell derived extracellular vesicles in the in vitro 3-dimensional collagen system and showed an increasing signal with an increase in collagen concentration. Overall, microcalcification development accounted for ¹⁸F-fluoride PET signal in PET-positive, CT-negative regions of human atherosclerotic plaques, and high surface area in regions of small microcalcifications could amplify PET signal.