Although cardiovascular diseases are the leading cause of death globally, non-invasive and inexpensive diagnostic tools for the identification of associated unstable atherosclerotic plaques are not yet available. Scanning acoustic microscopy offers a high potential to fill this critical gap in patient care. However, convincing validation and calibration of this technique requires high resolution maps of Ca concentrations of atherosclerotic plaques. Here, we demonstrate that synchrotron radiation-based X-ray fluorescence imaging with micrometer spatial resolution can provide such a gold standard.