Coupled X‑ray Fluorescence and X‑ray Absorption Spectroscopy for Microscale Imaging and Identification of Sulfur Species within Tissues and Skeletons of Scleractinian Corals

Identifying and mapping the wide range of sulfur species within complex matrices presents a challenge for understanding the distribution of these important biomolecules within environmental and biological systems. Here, we present a coupled micro X-ray fluorescence (μXRF) and X-ray absorption near-edge structure (XANES) spectroscopy method for determining the presence of specific sulfur species in coral tissues and skeletons at high spatial resolution. By using multiple energy stacks and principal component analysis of a large spectral database, we were able to more accurately identify sulfur species components and distinguish different species and distributions of sulfur formerly unresolved by previous studies. Specifically, coral tissues were dominated by more reduced sulfur species, such as glutathione disulfide, cysteine, and sulfoxide, as well as organic sulfate as represented by chondroitin sulfate. Sulfoxide distributions were visually correlated with the presence of zooxanthellae endosymbionts. Coral skeletons were composed primarily of carbonate-associated sulfate (CAS) along with minor contributions from organic sulfate and a separate inorganic sulfate likely in the form of adsorbed sulfate. This coupled XRF-XANES approach allows for a more accurate and informative view of sulfur within biological systems in situ and holds great promise for pairing with other techniques to allow for a more encompassing understanding of elemental distributions within the environment