For the past several decades, synchrotron radiation has been extensively used to measure the spatial distribution and chemical affinity of elements found in trace concentrations (<few µg/g) in animal and human tissues. Intense and highly focused (lateral size of several micrometers) X-ray beams combined with small steps of photon energy tuning (2–3 eV) of synchrotron radiation allowed X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) techniques to nondestructively and simultaneously detect trace elements as well as identify their chemical affinity and speciation in situ, respectively. Although limited by measurement time and radiation damage to the tissue, these techniques are commonly used to obtain two-dimensional and three-dimensional maps of several elements at synchrotron facilities around the world. The spatial distribution and chemistry of the trace elements obtained is then correlated to the targeted anatomical structures and to the biological functions (normal or pathological). For example, synchrotron-based in vitro studies of various human tissues showed significant differences between the normal and pathological distributions of metallic trace elements such as iron, zinc, copper, and lead in relation to human diseases ranging from Parkinson’s disease and cancer to osteoporosis and osteoarthritis. Current research effort is aimed at not only measuring the abnormal elemental distributions associated with various diseases, but also indicate or discover possible biological mechanisms that could explain such observations. While a number of studies confirmed and strengthened previous knowledge, others revealed or suggested new possible roles of trace elements or provided a more accurate spatial distribution in relation to the underlying histology. This area of research is at the intersection of several current fundamental and applied scientific inquiries such as metabolomics, medicine, biochemistry, toxicology, food science, health physics, and environmental and public health.