A Correlative X‑ray Bioimaging Triad for Metals in Biomedical Research

Metals are essential for numerous biological processes. They are widespread in tissues and cells. Advances in understanding the roles of the ten or more essential metals in health and disease would be accelerated by elucidation of their distribution and speciation in situ, and similarly, the development of metallodrugs. Here, we illustrate how advances in biochemical imaging can reveal the spatial distribution, oxidation state, and coordination environment of metals in biological systems and contribute to understanding both metal function and the mechanistic fate of metal-based therapeutics. Synchrotron-based X-ray techniques offer powerful tools for chemical and biochemical imaging at the cellular and subcellular levels by probing metals in cells and tissues under near-native conditions. The single-, or preferably, multimodal, use of the triad of X-ray fluorescence (XRF) mapping, X-ray absorption spectroscopy (XAS), and cryogenic soft X-ray tomography (cryo-SXT) for imaging allows the heterogeneity of cells and tissues to be studied, and also offers comprehensive element-specific insights into metal transformations in biological media. XRF allows nondestructive, multielemental imaging with sub-50 nm spatial resolution, while XAS provides site-specific electronic and structural speciation. Cryo-SXT offers three-dimensional ultrastructural imaging at ∼40 nm resolution by exploiting differential absorption in the water-window, preserving cellular architecture without the need for chemical fixation. Key considerations of emerging workflows that support cryogenic imaging and highlight correlative imaging strategies combining XRF, XAS, and SXT using compatible sample platforms and sample preparation, including cryo-fixation and freeze-drying, are discussed in relation to their impact on measurements. The convergence of these techniques under integrated, cryo-preserved conditions provides potential future breakthroughs in drug development and disease pathogenesis. Continued innovation in synchrotron optics, detector sensitivity, and sample environments will advance the implementation of this correlative X-ray bioimaging triad in biomedical research.