Bridging the Gap Between Micro- and Nanoscale Chemical Imaging of Biomaterials: a Multimodal and Multiscale Characterization Framework.

Understanding biomineralization processes requires experimental strategies that can correlate morphological, chemical, and structural information across scales. However, bridging microscale morphological and chemical descriptions with nanoscale molecular information remains a major experimental challenge. The multimodal and multiscale workflow developed in this work integrates scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDX), infrared microspectroscopy (μFT-IR), and atomic force microscopy-infrared spectroscopy (AFM-IR) performed sequentially on the same specimen. It addresses the main challenges associated with multimodal analysis, including sample preparation compatibility, measurement chronology, and region-of-interest colocalization through a straightforward colocalization and image registration procedure. When applied to a breast biopsy sample containing microcalcifications, this experimental strategy enabled the resolution of both inter- and intra-deposit chemical heterogeneities. By integrating complementary spectroscopy datasets, this workflow provides a chemical description of mineralized tissues spanning the micro- to the nanoscale, thereby linking the specimen's global and local physicochemical properties. Beyond this application, this study provides a methodological framework for investigating mineralization pathways and hierarchical organization in complex biomineralized systems.